<?xml version="1.0" encoding="US-ASCII" standalone="no"?>
<!--Generated by ThunderDome XBRL - 03:20PM UTC 2026-05-12--><link:linkbase xmlns:link="http://www.xbrl.org/2003/linkbase" xmlns:xbrldt="http://xbrl.org/2005/xbrldt" xmlns:xlink="http://www.w3.org/1999/xlink" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" xsi:schemaLocation="http://www.xbrl.org/2003/linkbase http://www.xbrl.org/2003/xbrl-linkbase-2003-12-31.xsd">
  <link:arcroleRef arcroleURI="http://xbrl.org/int/dim/arcrole/all" xlink:href="http://www.xbrl.org/2005/xbrldt-2005.xsd#all" xlink:type="simple"/>
  <link:arcroleRef arcroleURI="http://xbrl.org/int/dim/arcrole/domain-member" xlink:href="http://www.xbrl.org/2005/xbrldt-2005.xsd#domain-member" xlink:type="simple"/>
  <link:arcroleRef arcroleURI="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:href="http://www.xbrl.org/2005/xbrldt-2005.xsd#dimension-domain" xlink:type="simple"/>
  <link:arcroleRef arcroleURI="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:href="http://www.xbrl.org/2005/xbrldt-2005.xsd#hypercube-dimension" xlink:type="simple"/>
  <link:arcroleRef arcroleURI="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:href="http://www.xbrl.org/2005/xbrldt-2005.xsd#dimension-default" xlink:type="simple"/>
  <link:roleRef roleURI="http://www.talphera.com/20260331/role/statement-note-5-commitments-and-contingencies" xlink:href="tlph-20260331.xsd#statement-note-5-commitments-and-contingencies" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://www.talphera.com/20260331/role/statement-note-5-commitments-and-contingencies" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_CommitmentsAndContingenciesDisclosureTextBlock" xlink:label="us-gaap_CommitmentsAndContingenciesDisclosureTextBlock" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DisclosureTextBlockAbstract" xlink:label="us-gaap_DisclosureTextBlockAbstract" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_LossContingenciesTable" xlink:label="us-gaap_LossContingenciesTable" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DisclosureTextBlockAbstract" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_LossContingenciesTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_LossContingenciesTable" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DisclosureTextBlockAbstract" xlink:to="us-gaap_CommitmentsAndContingenciesDisclosureTextBlock" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.talphera.com/20260331/role/statement-document-and-entity-information" xlink:href="tlph-20260331.xsd#statement-document-and-entity-information" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://www.talphera.com/20260331/role/statement-document-and-entity-information" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_AmendmentFlag" xlink:label="dei_AmendmentFlag" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_CityAreaCode" xlink:label="dei_CityAreaCode" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_CurrentFiscalYearEndDate" xlink:label="dei_CurrentFiscalYearEndDate" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_DocumentFiscalPeriodFocus" xlink:label="dei_DocumentFiscalPeriodFocus" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_DocumentFiscalYearFocus" xlink:label="dei_DocumentFiscalYearFocus" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_DocumentInformationLineItems" xlink:label="dei_DocumentInformationLineItems" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_DocumentInformationTable" xlink:label="dei_DocumentInformationTable" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_DocumentPeriodEndDate" xlink:label="dei_DocumentPeriodEndDate" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_DocumentQuarterlyReport" xlink:label="dei_DocumentQuarterlyReport" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_DocumentTransitionReport" xlink:label="dei_DocumentTransitionReport" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_DocumentType" xlink:label="dei_DocumentType" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityAddressAddressLine1" xlink:label="dei_EntityAddressAddressLine1" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityAddressCityOrTown" xlink:label="dei_EntityAddressCityOrTown" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityAddressPostalZipCode" xlink:label="dei_EntityAddressPostalZipCode" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityAddressStateOrProvince" xlink:label="dei_EntityAddressStateOrProvince" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityCentralIndexKey" xlink:label="dei_EntityCentralIndexKey" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityCommonStockSharesOutstanding" xlink:label="dei_EntityCommonStockSharesOutstanding" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityCurrentReportingStatus" xlink:label="dei_EntityCurrentReportingStatus" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityEmergingGrowthCompany" xlink:label="dei_EntityEmergingGrowthCompany" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityFileNumber" xlink:label="dei_EntityFileNumber" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityFilerCategory" xlink:label="dei_EntityFilerCategory" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityIncorporationStateCountryCode" xlink:label="dei_EntityIncorporationStateCountryCode" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityInteractiveDataCurrent" xlink:label="dei_EntityInteractiveDataCurrent" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityRegistrantName" xlink:label="dei_EntityRegistrantName" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityShellCompany" xlink:label="dei_EntityShellCompany" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntitySmallBusiness" xlink:label="dei_EntitySmallBusiness" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityTaxIdentificationNumber" xlink:label="dei_EntityTaxIdentificationNumber" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_LocalPhoneNumber" xlink:label="dei_LocalPhoneNumber" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_Security12bTitle" xlink:label="dei_Security12bTitle" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_SecurityExchangeName" xlink:label="dei_SecurityExchangeName" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_TradingSymbol" xlink:label="dei_TradingSymbol" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="dei_DocumentInformationTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_DocumentInformationTable" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_DocumentType" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_DocumentQuarterlyReport" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_DocumentPeriodEndDate" xlink:type="arc"/>
    <link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_DocumentTransitionReport" xlink:type="arc"/>
    <link:definitionArc order="5" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_EntityFileNumber" xlink:type="arc"/>
    <link:definitionArc order="6" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_EntityRegistrantName" xlink:type="arc"/>
    <link:definitionArc order="7" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_EntityIncorporationStateCountryCode" xlink:type="arc"/>
    <link:definitionArc order="8" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_EntityTaxIdentificationNumber" xlink:type="arc"/>
    <link:definitionArc order="9" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_EntityAddressAddressLine1" xlink:type="arc"/>
    <link:definitionArc order="10" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_EntityAddressCityOrTown" xlink:type="arc"/>
    <link:definitionArc order="11" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_EntityAddressStateOrProvince" xlink:type="arc"/>
    <link:definitionArc order="12" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_EntityAddressPostalZipCode" xlink:type="arc"/>
    <link:definitionArc order="13" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_CityAreaCode" xlink:type="arc"/>
    <link:definitionArc order="14" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_LocalPhoneNumber" xlink:type="arc"/>
    <link:definitionArc order="15" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_Security12bTitle" xlink:type="arc"/>
    <link:definitionArc order="16" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_TradingSymbol" xlink:type="arc"/>
    <link:definitionArc order="17" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_SecurityExchangeName" xlink:type="arc"/>
    <link:definitionArc order="18" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_EntityCurrentReportingStatus" xlink:type="arc"/>
    <link:definitionArc order="19" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_EntityInteractiveDataCurrent" xlink:type="arc"/>
    <link:definitionArc order="20" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_EntityFilerCategory" xlink:type="arc"/>
    <link:definitionArc order="21" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_EntitySmallBusiness" xlink:type="arc"/>
    <link:definitionArc order="22" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_EntityEmergingGrowthCompany" xlink:type="arc"/>
    <link:definitionArc order="23" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_EntityShellCompany" xlink:type="arc"/>
    <link:definitionArc order="24" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_EntityCommonStockSharesOutstanding" xlink:type="arc"/>
    <link:definitionArc order="25" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_EntityCentralIndexKey" xlink:type="arc"/>
    <link:definitionArc order="26" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_CurrentFiscalYearEndDate" xlink:type="arc"/>
    <link:definitionArc order="27" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_DocumentFiscalYearFocus" xlink:type="arc"/>
    <link:definitionArc order="28" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_DocumentFiscalPeriodFocus" xlink:type="arc"/>
    <link:definitionArc order="29" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_AmendmentFlag" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.talphera.com/20260331/role/statement--consolidated-balance-sheets-unaudited-" xlink:href="tlph-20260331.xsd#statement--consolidated-balance-sheets-unaudited-" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://www.talphera.com/20260331/role/statement--consolidated-balance-sheets-unaudited-" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="tlph-20260331.xsd#tlph_LiabilitiesRelatedToTheSaleOfFuturePayments" xlink:label="tlph_LiabilitiesRelatedToTheSaleOfFuturePayments" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AccountsPayableCurrent" xlink:label="us-gaap_AccountsPayableCurrent" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AccruedLiabilitiesCurrent" xlink:label="us-gaap_AccruedLiabilitiesCurrent" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AdditionalPaidInCapital" xlink:label="us-gaap_AdditionalPaidInCapital" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_Assets" xlink:label="us-gaap_Assets" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AssetsCurrent" xlink:label="us-gaap_AssetsCurrent" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AssetsCurrentAbstract" xlink:label="us-gaap_AssetsCurrentAbstract" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_CashAndCashEquivalentsAtCarryingValue" xlink:label="us-gaap_CashAndCashEquivalentsAtCarryingValue" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_CommitmentsAndContingencies" xlink:label="us-gaap_CommitmentsAndContingencies" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_CommonStockValue" xlink:label="us-gaap_CommonStockValue" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_IntangibleAssetsNetExcludingGoodwill" xlink:label="us-gaap_IntangibleAssetsNetExcludingGoodwill" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_Liabilities" xlink:label="us-gaap_Liabilities" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_LiabilitiesAndStockholdersEquity" xlink:label="us-gaap_LiabilitiesAndStockholdersEquity" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_LiabilitiesCurrent" xlink:label="us-gaap_LiabilitiesCurrent" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_LiabilitiesCurrentAbstract" xlink:label="us-gaap_LiabilitiesCurrentAbstract" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_LiabilitiesOfDisposalGroupIncludingDiscontinuedOperationCurrent" xlink:label="us-gaap_LiabilitiesOfDisposalGroupIncludingDiscontinuedOperationCurrent" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_PrepaidExpenseAndOtherAssetsCurrent" xlink:label="us-gaap_PrepaidExpenseAndOtherAssetsCurrent" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_RetainedEarningsAccumulatedDeficit" xlink:label="us-gaap_RetainedEarningsAccumulatedDeficit" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShortTermInvestments" xlink:label="us-gaap_ShortTermInvestments" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StockholdersEquity" xlink:label="us-gaap_StockholdersEquity" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StockholdersEquityAbstract" xlink:label="us-gaap_StockholdersEquityAbstract" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_WarrantsAndRightsOutstanding" xlink:label="us-gaap_WarrantsAndRightsOutstanding" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_AssetsCurrentAbstract" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AssetsCurrentAbstract" xlink:to="us-gaap_CashAndCashEquivalentsAtCarryingValue" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AssetsCurrentAbstract" xlink:to="us-gaap_ShortTermInvestments" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AssetsCurrentAbstract" xlink:to="us-gaap_PrepaidExpenseAndOtherAssetsCurrent" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AssetsCurrentAbstract" xlink:to="us-gaap_AssetsCurrent" xlink:type="arc"/>
    <link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AssetsCurrentAbstract" xlink:to="us-gaap_IntangibleAssetsNetExcludingGoodwill" xlink:type="arc"/>
    <link:definitionArc order="5" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AssetsCurrentAbstract" xlink:to="us-gaap_Assets" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_LiabilitiesCurrentAbstract" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_LiabilitiesCurrentAbstract" xlink:to="us-gaap_AccountsPayableCurrent" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_LiabilitiesCurrentAbstract" xlink:to="us-gaap_AccruedLiabilitiesCurrent" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_LiabilitiesCurrentAbstract" xlink:to="us-gaap_LiabilitiesOfDisposalGroupIncludingDiscontinuedOperationCurrent" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_LiabilitiesCurrentAbstract" xlink:to="us-gaap_LiabilitiesCurrent" xlink:type="arc"/>
    <link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_LiabilitiesCurrentAbstract" xlink:to="us-gaap_WarrantsAndRightsOutstanding" xlink:type="arc"/>
    <link:definitionArc order="5" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_LiabilitiesCurrentAbstract" xlink:to="tlph_LiabilitiesRelatedToTheSaleOfFuturePayments" xlink:type="arc"/>
    <link:definitionArc order="6" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_LiabilitiesCurrentAbstract" xlink:to="us-gaap_Liabilities" xlink:type="arc"/>
    <link:definitionArc order="7" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_LiabilitiesCurrentAbstract" xlink:to="us-gaap_CommitmentsAndContingencies" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StockholdersEquityAbstract" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StockholdersEquityAbstract" xlink:to="us-gaap_CommonStockValue" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StockholdersEquityAbstract" xlink:to="us-gaap_AdditionalPaidInCapital" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StockholdersEquityAbstract" xlink:to="us-gaap_RetainedEarningsAccumulatedDeficit" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StockholdersEquityAbstract" xlink:to="us-gaap_StockholdersEquity" xlink:type="arc"/>
    <link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StockholdersEquityAbstract" xlink:to="us-gaap_LiabilitiesAndStockholdersEquity" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.talphera.com/20260331/role/statement--consolidated-balance-sheets-unaudited-parentheticals" xlink:href="tlph-20260331.xsd#statement--consolidated-balance-sheets-unaudited-parentheticals" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://www.talphera.com/20260331/role/statement--consolidated-balance-sheets-unaudited-parentheticals" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_CommonStockParOrStatedValuePerShare" xlink:label="us-gaap_CommonStockParOrStatedValuePerShare" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_CommonStockSharesAuthorized" xlink:label="us-gaap_CommonStockSharesAuthorized" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_CommonStockSharesIssued" xlink:label="us-gaap_CommonStockSharesIssued" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_CommonStockSharesOutstanding" xlink:label="us-gaap_CommonStockSharesOutstanding" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_CommonStockParOrStatedValuePerShare" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_CommonStockSharesAuthorized" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_CommonStockSharesIssued" xlink:type="arc"/>
    <link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_CommonStockSharesOutstanding" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.talphera.com/20260331/role/statement-consolidated-statements-of-operations-unaudited" xlink:href="tlph-20260331.xsd#statement-consolidated-statements-of-operations-unaudited" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://www.talphera.com/20260331/role/statement-consolidated-statements-of-operations-unaudited" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_CostsAndExpenses" xlink:label="us-gaap_CostsAndExpenses" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_EarningsPerShareAbstract" xlink:label="us-gaap_EarningsPerShareAbstract" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_EarningsPerShareBasic" xlink:label="us-gaap_EarningsPerShareBasic" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_FairValueAdjustmentOfWarrants" xlink:label="us-gaap_FairValueAdjustmentOfWarrants" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_IncomeLossFromContinuingOperations" xlink:label="us-gaap_IncomeLossFromContinuingOperations" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_IncomeLossFromContinuingOperationsPerBasicShare" xlink:label="us-gaap_IncomeLossFromContinuingOperationsPerBasicShare" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_IncomeLossFromDiscontinuedOperationsNetOfTaxAttributableToReportingEntity" xlink:label="us-gaap_IncomeLossFromDiscontinuedOperationsNetOfTaxAttributableToReportingEntity" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_IncomeLossFromDiscontinuedOperationsNetOfTaxPerBasicShare" xlink:label="us-gaap_IncomeLossFromDiscontinuedOperationsNetOfTaxPerBasicShare" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_InvestmentIncomeNet" xlink:label="us-gaap_InvestmentIncomeNet" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_NetIncomeLoss" xlink:label="us-gaap_NetIncomeLoss" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_NonoperatingIncomeExpense" xlink:label="us-gaap_NonoperatingIncomeExpense" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_NonoperatingIncomeExpenseAbstract" xlink:label="us-gaap_NonoperatingIncomeExpenseAbstract" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_OperatingCostsAndExpensesAbstract" xlink:label="us-gaap_OperatingCostsAndExpensesAbstract" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_OperatingIncomeLoss" xlink:label="us-gaap_OperatingIncomeLoss" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ResearchAndDevelopmentExpense" xlink:label="us-gaap_ResearchAndDevelopmentExpense" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_RevenueFromContractWithCustomerIncludingAssessedTax" xlink:label="us-gaap_RevenueFromContractWithCustomerIncludingAssessedTax" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SellingGeneralAndAdministrativeExpense" xlink:label="us-gaap_SellingGeneralAndAdministrativeExpense" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_WeightedAverageNumberOfSharesOutstandingBasic" xlink:label="us-gaap_WeightedAverageNumberOfSharesOutstandingBasic" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_RevenueFromContractWithCustomerIncludingAssessedTax" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_OperatingCostsAndExpensesAbstract" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_OperatingCostsAndExpensesAbstract" xlink:to="us-gaap_ResearchAndDevelopmentExpense" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_OperatingCostsAndExpensesAbstract" xlink:to="us-gaap_SellingGeneralAndAdministrativeExpense" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_OperatingCostsAndExpensesAbstract" xlink:to="us-gaap_CostsAndExpenses" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_OperatingCostsAndExpensesAbstract" xlink:to="us-gaap_OperatingIncomeLoss" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_NonoperatingIncomeExpenseAbstract" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_NonoperatingIncomeExpenseAbstract" xlink:to="us-gaap_InvestmentIncomeNet" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_NonoperatingIncomeExpenseAbstract" xlink:to="us-gaap_FairValueAdjustmentOfWarrants" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_NonoperatingIncomeExpenseAbstract" xlink:to="us-gaap_NonoperatingIncomeExpense" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_NonoperatingIncomeExpenseAbstract" xlink:to="us-gaap_IncomeLossFromContinuingOperations" xlink:type="arc"/>
    <link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_NonoperatingIncomeExpenseAbstract" xlink:to="us-gaap_IncomeLossFromDiscontinuedOperationsNetOfTaxAttributableToReportingEntity" xlink:type="arc"/>
    <link:definitionArc order="5" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_NonoperatingIncomeExpenseAbstract" xlink:to="us-gaap_NetIncomeLoss" xlink:type="arc"/>
    <link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_EarningsPerShareAbstract" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_EarningsPerShareAbstract" xlink:to="us-gaap_IncomeLossFromContinuingOperationsPerBasicShare" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_EarningsPerShareAbstract" xlink:to="us-gaap_IncomeLossFromDiscontinuedOperationsNetOfTaxPerBasicShare" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_EarningsPerShareAbstract" xlink:to="us-gaap_EarningsPerShareBasic" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_EarningsPerShareAbstract" xlink:to="us-gaap_WeightedAverageNumberOfSharesOutstandingBasic" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.talphera.com/20260331/role/statement-consolidated-statements-of-stockholders-equity-unaudited" xlink:href="tlph-20260331.xsd#statement-consolidated-statements-of-stockholders-equity-unaudited" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://www.talphera.com/20260331/role/statement-consolidated-statements-of-stockholders-equity-unaudited" xlink:type="extended">
    <link:loc xlink:href="tlph-20260331.xsd#tlph_CommonStockAndWarrantsIssuedDuringPeriodSharesNewIssues" xlink:label="tlph_CommonStockAndWarrantsIssuedDuringPeriodSharesNewIssues" xlink:type="locator"/>
    <link:loc xlink:href="tlph-20260331.xsd#tlph_CommonStockAndWarrantsIssuedDuringPeriodValueNewIssues" xlink:label="tlph_CommonStockAndWarrantsIssuedDuringPeriodValueNewIssues" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AdditionalPaidInCapitalMember" xlink:label="us-gaap_AdditionalPaidInCapitalMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AdjustmentsToAdditionalPaidInCapitalSharebasedCompensationRequisiteServicePeriodRecognitionValue" xlink:label="us-gaap_AdjustmentsToAdditionalPaidInCapitalSharebasedCompensationRequisiteServicePeriodRecognitionValue" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_CommonStockMember" xlink:label="us-gaap_CommonStockMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_EquityComponentDomain" xlink:label="us-gaap_EquityComponentDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_EquityComponentDomain" xlink:label="us-gaap_EquityComponentDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_NetIncomeLoss" xlink:label="us-gaap_NetIncomeLoss" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_RetainedEarningsMember" xlink:label="us-gaap_RetainedEarningsMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SharesOutstanding" xlink:label="us-gaap_SharesOutstanding" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementEquityComponentsAxis" xlink:label="us-gaap_StatementEquityComponentsAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StockIssuedDuringPeriodSharesEmployeeStockPurchasePlans" xlink:label="us-gaap_StockIssuedDuringPeriodSharesEmployeeStockPurchasePlans" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StockIssuedDuringPeriodSharesRestrictedStockAwardNetOfForfeitures" xlink:label="us-gaap_StockIssuedDuringPeriodSharesRestrictedStockAwardNetOfForfeitures" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StockIssuedDuringPeriodValueEmployeeStockPurchasePlan" xlink:label="us-gaap_StockIssuedDuringPeriodValueEmployeeStockPurchasePlan" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StockIssuedDuringPeriodValueRestrictedStockAwardNetOfForfeitures" xlink:label="us-gaap_StockIssuedDuringPeriodValueRestrictedStockAwardNetOfForfeitures" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StockholdersEquity" xlink:label="us-gaap_StockholdersEquity" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_StatementEquityComponentsAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_EquityComponentDomain" xlink:to="us-gaap_CommonStockMember" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_StatementEquityComponentsAxis" xlink:to="us-gaap_EquityComponentDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_EquityComponentDomain" xlink:to="us-gaap_AdditionalPaidInCapitalMember" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_EquityComponentDomain" xlink:to="us-gaap_RetainedEarningsMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_StatementEquityComponentsAxis" xlink:to="us-gaap_EquityComponentDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_SharesOutstanding" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StockholdersEquity" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_AdjustmentsToAdditionalPaidInCapitalSharebasedCompensationRequisiteServicePeriodRecognitionValue" xlink:type="arc"/>
    <link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StockIssuedDuringPeriodSharesRestrictedStockAwardNetOfForfeitures" xlink:type="arc"/>
    <link:definitionArc order="5" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StockIssuedDuringPeriodValueRestrictedStockAwardNetOfForfeitures" xlink:type="arc"/>
    <link:definitionArc order="6" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="tlph_CommonStockAndWarrantsIssuedDuringPeriodSharesNewIssues" xlink:type="arc"/>
    <link:definitionArc order="7" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="tlph_CommonStockAndWarrantsIssuedDuringPeriodValueNewIssues" xlink:type="arc"/>
    <link:definitionArc order="8" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StockIssuedDuringPeriodSharesEmployeeStockPurchasePlans" xlink:type="arc"/>
    <link:definitionArc order="9" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StockIssuedDuringPeriodValueEmployeeStockPurchasePlan" xlink:type="arc"/>
    <link:definitionArc order="10" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_NetIncomeLoss" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.talphera.com/20260331/role/statement-condensed-consolidated-statements-of-cash-flows-unaudited" xlink:href="tlph-20260331.xsd#statement-condensed-consolidated-statements-of-cash-flows-unaudited" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://www.talphera.com/20260331/role/statement-condensed-consolidated-statements-of-cash-flows-unaudited" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="tlph-20260331.xsd#tlph_AmortizationOfDiscountOnShortTermInvestments" xlink:label="tlph_AmortizationOfDiscountOnShortTermInvestments" xlink:type="locator"/>
    <link:loc xlink:href="tlph-20260331.xsd#tlph_Prefunded2024WarrantsMember" xlink:label="tlph_Prefunded2024WarrantsMember" xlink:type="locator"/>
    <link:loc xlink:href="tlph-20260331.xsd#tlph_ProceedsPaymentsIssuanceOfSharesShareBasedPaymentArrangementIncludingOptionExercised" xlink:label="tlph_ProceedsPaymentsIssuanceOfSharesShareBasedPaymentArrangementIncludingOptionExercised" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AdjustmentsToReconcileNetIncomeLossToCashProvidedByUsedInOperatingActivitiesAbstract" xlink:label="us-gaap_AdjustmentsToReconcileNetIncomeLossToCashProvidedByUsedInOperatingActivitiesAbstract" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_CashCashEquivalentsRestrictedCashAndRestrictedCashEquivalentsIncludingDisposalGroupAndDiscontinuedOperations" xlink:label="us-gaap_CashCashEquivalentsRestrictedCashAndRestrictedCashEquivalentsIncludingDisposalGroupAndDiscontinuedOperations" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_CashCashEquivalentsRestrictedCashAndRestrictedCashEquivalentsPeriodIncreaseDecreaseIncludingExchangeRateEffect" xlink:label="us-gaap_CashCashEquivalentsRestrictedCashAndRestrictedCashEquivalentsPeriodIncreaseDecreaseIncludingExchangeRateEffect" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_FairValueAdjustmentOfWarrants" xlink:label="us-gaap_FairValueAdjustmentOfWarrants" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_IncreaseDecreaseInAccountsPayable" xlink:label="us-gaap_IncreaseDecreaseInAccountsPayable" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_IncreaseDecreaseInAccountsReceivable" xlink:label="us-gaap_IncreaseDecreaseInAccountsReceivable" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_IncreaseDecreaseInAccruedLiabilities" xlink:label="us-gaap_IncreaseDecreaseInAccruedLiabilities" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_IncreaseDecreaseInOperatingAssetsAbstract" xlink:label="us-gaap_IncreaseDecreaseInOperatingAssetsAbstract" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_IncreaseDecreaseInPrepaidDeferredExpenseAndOtherAssets" xlink:label="us-gaap_IncreaseDecreaseInPrepaidDeferredExpenseAndOtherAssets" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_NetCashProvidedByUsedInFinancingActivities" xlink:label="us-gaap_NetCashProvidedByUsedInFinancingActivities" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_NetCashProvidedByUsedInFinancingActivitiesAbstract" xlink:label="us-gaap_NetCashProvidedByUsedInFinancingActivitiesAbstract" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_NetCashProvidedByUsedInInvestingActivities" xlink:label="us-gaap_NetCashProvidedByUsedInInvestingActivities" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_NetCashProvidedByUsedInInvestingActivitiesAbstract" xlink:label="us-gaap_NetCashProvidedByUsedInInvestingActivitiesAbstract" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_NetCashProvidedByUsedInOperatingActivities" xlink:label="us-gaap_NetCashProvidedByUsedInOperatingActivities" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_NetCashProvidedByUsedInOperatingActivitiesAbstract" xlink:label="us-gaap_NetCashProvidedByUsedInOperatingActivitiesAbstract" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_NetIncomeLoss" xlink:label="us-gaap_NetIncomeLoss" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_PaymentsToAcquireShortTermInvestments" xlink:label="us-gaap_PaymentsToAcquireShortTermInvestments" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ProceedsFromIssuanceOfWarrants" xlink:label="us-gaap_ProceedsFromIssuanceOfWarrants" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ProceedsFromSaleMaturityAndCollectionsOfInvestments" xlink:label="us-gaap_ProceedsFromSaleMaturityAndCollectionsOfInvestments" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SaleOfStockNameOfTransactionDomain" xlink:label="us-gaap_SaleOfStockNameOfTransactionDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SaleOfStockNameOfTransactionDomain" xlink:label="us-gaap_SaleOfStockNameOfTransactionDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensation" xlink:label="us-gaap_ShareBasedCompensation" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SubsidiarySaleOfStockAxis" xlink:label="us-gaap_SubsidiarySaleOfStockAxis" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_SubsidiarySaleOfStockAxis" xlink:to="us-gaap_SaleOfStockNameOfTransactionDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_SubsidiarySaleOfStockAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_SaleOfStockNameOfTransactionDomain" xlink:to="tlph_Prefunded2024WarrantsMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_SubsidiarySaleOfStockAxis" xlink:to="us-gaap_SaleOfStockNameOfTransactionDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_NetCashProvidedByUsedInOperatingActivitiesAbstract" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_NetCashProvidedByUsedInOperatingActivitiesAbstract" xlink:to="us-gaap_NetIncomeLoss" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_AdjustmentsToReconcileNetIncomeLossToCashProvidedByUsedInOperatingActivitiesAbstract" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AdjustmentsToReconcileNetIncomeLossToCashProvidedByUsedInOperatingActivitiesAbstract" xlink:to="tlph_AmortizationOfDiscountOnShortTermInvestments" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AdjustmentsToReconcileNetIncomeLossToCashProvidedByUsedInOperatingActivitiesAbstract" xlink:to="us-gaap_ShareBasedCompensation" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AdjustmentsToReconcileNetIncomeLossToCashProvidedByUsedInOperatingActivitiesAbstract" xlink:to="us-gaap_FairValueAdjustmentOfWarrants" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_IncreaseDecreaseInOperatingAssetsAbstract" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_IncreaseDecreaseInOperatingAssetsAbstract" xlink:to="us-gaap_IncreaseDecreaseInAccountsReceivable" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_IncreaseDecreaseInOperatingAssetsAbstract" xlink:to="us-gaap_IncreaseDecreaseInPrepaidDeferredExpenseAndOtherAssets" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_IncreaseDecreaseInOperatingAssetsAbstract" xlink:to="us-gaap_IncreaseDecreaseInAccountsPayable" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_IncreaseDecreaseInOperatingAssetsAbstract" xlink:to="us-gaap_IncreaseDecreaseInAccruedLiabilities" xlink:type="arc"/>
    <link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_IncreaseDecreaseInOperatingAssetsAbstract" xlink:to="us-gaap_NetCashProvidedByUsedInOperatingActivities" xlink:type="arc"/>
    <link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_NetCashProvidedByUsedInInvestingActivitiesAbstract" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_NetCashProvidedByUsedInInvestingActivitiesAbstract" xlink:to="us-gaap_PaymentsToAcquireShortTermInvestments" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_NetCashProvidedByUsedInInvestingActivitiesAbstract" xlink:to="us-gaap_ProceedsFromSaleMaturityAndCollectionsOfInvestments" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_NetCashProvidedByUsedInInvestingActivitiesAbstract" xlink:to="us-gaap_NetCashProvidedByUsedInInvestingActivities" xlink:type="arc"/>
    <link:definitionArc order="5" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_NetCashProvidedByUsedInFinancingActivitiesAbstract" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_NetCashProvidedByUsedInFinancingActivitiesAbstract" xlink:to="us-gaap_ProceedsFromIssuanceOfWarrants" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_NetCashProvidedByUsedInFinancingActivitiesAbstract" xlink:to="tlph_ProceedsPaymentsIssuanceOfSharesShareBasedPaymentArrangementIncludingOptionExercised" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_NetCashProvidedByUsedInFinancingActivitiesAbstract" xlink:to="us-gaap_NetCashProvidedByUsedInFinancingActivities" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_NetCashProvidedByUsedInFinancingActivitiesAbstract" xlink:to="us-gaap_CashCashEquivalentsRestrictedCashAndRestrictedCashEquivalentsPeriodIncreaseDecreaseIncludingExchangeRateEffect" xlink:type="arc"/>
    <link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_NetCashProvidedByUsedInFinancingActivitiesAbstract" xlink:to="us-gaap_CashCashEquivalentsRestrictedCashAndRestrictedCashEquivalentsIncludingDisposalGroupAndDiscontinuedOperations" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.talphera.com/20260331/role/statement-note-1-organization-and-summary-of-significant-accounting-policies" xlink:href="tlph-20260331.xsd#statement-note-1-organization-and-summary-of-significant-accounting-policies" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://www.talphera.com/20260331/role/statement-note-1-organization-and-summary-of-significant-accounting-policies" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_BasisOfPresentationAndSignificantAccountingPoliciesTextBlock" xlink:label="us-gaap_BasisOfPresentationAndSignificantAccountingPoliciesTextBlock" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DisclosureTextBlockAbstract" xlink:label="us-gaap_DisclosureTextBlockAbstract" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ScheduleOfProductInformationTable" xlink:label="us-gaap_ScheduleOfProductInformationTable" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DisclosureTextBlockAbstract" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_ScheduleOfProductInformationTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ScheduleOfProductInformationTable" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DisclosureTextBlockAbstract" xlink:to="us-gaap_BasisOfPresentationAndSignificantAccountingPoliciesTextBlock" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.talphera.com/20260331/role/statement-note-2-investments-and-fair-value-measurement" xlink:href="tlph-20260331.xsd#statement-note-2-investments-and-fair-value-measurement" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://www.talphera.com/20260331/role/statement-note-2-investments-and-fair-value-measurement" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="tlph-20260331.xsd#tlph_CommonWarrantsMember" xlink:label="tlph_CommonWarrantsMember" xlink:type="locator"/>
    <link:loc xlink:href="tlph-20260331.xsd#tlph_InvestmentsAndFairValueMeasurementDisclosureTextBlock" xlink:label="tlph_InvestmentsAndFairValueMeasurementDisclosureTextBlock" xlink:type="locator"/>
    <link:loc xlink:href="tlph-20260331.xsd#tlph_MarketableSecuritiesMember" xlink:label="tlph_MarketableSecuritiesMember" xlink:type="locator"/>
    <link:loc xlink:href="tlph-20260331.xsd#tlph_WarrantLiabilityMember" xlink:label="tlph_WarrantLiabilityMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_CashAndCashEquivalentsAxis" xlink:label="us-gaap_CashAndCashEquivalentsAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_CashAndCashEquivalentsMember" xlink:label="us-gaap_CashAndCashEquivalentsMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_CashMember" xlink:label="us-gaap_CashMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfWarrantOrRightAxis" xlink:label="us-gaap_ClassOfWarrantOrRightAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfWarrantOrRightDomain" xlink:label="us-gaap_ClassOfWarrantOrRightDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfWarrantOrRightDomain" xlink:label="us-gaap_ClassOfWarrantOrRightDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DisclosureTextBlockAbstract" xlink:label="us-gaap_DisclosureTextBlockAbstract" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisTable" xlink:label="us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisTable" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_FairValueAssetsMeasuredOnRecurringBasisUnobservableInputReconciliationByAssetClassDomain" xlink:label="us-gaap_FairValueAssetsMeasuredOnRecurringBasisUnobservableInputReconciliationByAssetClassDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_FairValueAssetsMeasuredOnRecurringBasisUnobservableInputReconciliationByAssetClassDomain" xlink:label="us-gaap_FairValueAssetsMeasuredOnRecurringBasisUnobservableInputReconciliationByAssetClassDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_FairValueByAssetClassAxis" xlink:label="us-gaap_FairValueByAssetClassAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_FairValueByFairValueHierarchyLevelAxis" xlink:label="us-gaap_FairValueByFairValueHierarchyLevelAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_FairValueInputsLevel1Member" xlink:label="us-gaap_FairValueInputsLevel1Member" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_FairValueInputsLevel2Member" xlink:label="us-gaap_FairValueInputsLevel2Member" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_FairValueInputsLevel3Member" xlink:label="us-gaap_FairValueInputsLevel3Member" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_FairValueMeasurementsFairValueHierarchyDomain" xlink:label="us-gaap_FairValueMeasurementsFairValueHierarchyDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_FairValueMeasurementsFairValueHierarchyDomain" xlink:label="us-gaap_FairValueMeasurementsFairValueHierarchyDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_FinancialInstrumentAxis" xlink:label="us-gaap_FinancialInstrumentAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_MeasurementInputExpectedDividendRateMember" xlink:label="us-gaap_MeasurementInputExpectedDividendRateMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_MeasurementInputExpectedTermMember" xlink:label="us-gaap_MeasurementInputExpectedTermMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_MeasurementInputPriceVolatilityMember" xlink:label="us-gaap_MeasurementInputPriceVolatilityMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_MeasurementInputRiskFreeInterestRateMember" xlink:label="us-gaap_MeasurementInputRiskFreeInterestRateMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_MeasurementInputSharePriceMember" xlink:label="us-gaap_MeasurementInputSharePriceMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_MeasurementInputTypeAxis" xlink:label="us-gaap_MeasurementInputTypeAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_MeasurementInputTypeDomain" xlink:label="us-gaap_MeasurementInputTypeDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_MeasurementInputTypeDomain" xlink:label="us-gaap_MeasurementInputTypeDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_MoneyMarketFundsMember" xlink:label="us-gaap_MoneyMarketFundsMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_RestrictedCashAndCashEquivalentsCashAndCashEquivalentsMember" xlink:label="us-gaap_RestrictedCashAndCashEquivalentsCashAndCashEquivalentsMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_RestrictedCashAndCashEquivalentsCashAndCashEquivalentsMember" xlink:label="us-gaap_RestrictedCashAndCashEquivalentsCashAndCashEquivalentsMember-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain" xlink:label="us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain" xlink:label="us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_USGovernmentAgenciesDebtSecuritiesMember" xlink:label="us-gaap_USGovernmentAgenciesDebtSecuritiesMember" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DisclosureTextBlockAbstract" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_ClassOfWarrantOrRightAxis" xlink:to="us-gaap_ClassOfWarrantOrRightDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_FairValueByAssetClassAxis" xlink:to="us-gaap_FairValueAssetsMeasuredOnRecurringBasisUnobservableInputReconciliationByAssetClassDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_FairValueByFairValueHierarchyLevelAxis" xlink:to="us-gaap_FairValueMeasurementsFairValueHierarchyDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_FinancialInstrumentAxis" xlink:to="us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_CashAndCashEquivalentsAxis" xlink:to="us-gaap_RestrictedCashAndCashEquivalentsCashAndCashEquivalentsMember" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_MeasurementInputTypeAxis" xlink:to="us-gaap_MeasurementInputTypeDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisTable" xlink:to="us-gaap_CashAndCashEquivalentsAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_RestrictedCashAndCashEquivalentsCashAndCashEquivalentsMember" xlink:to="us-gaap_CashAndCashEquivalentsMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_CashAndCashEquivalentsAxis" xlink:to="us-gaap_RestrictedCashAndCashEquivalentsCashAndCashEquivalentsMember-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisTable" xlink:to="us-gaap_FairValueByAssetClassAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_FairValueAssetsMeasuredOnRecurringBasisUnobservableInputReconciliationByAssetClassDomain" xlink:to="us-gaap_CashMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_FairValueByAssetClassAxis" xlink:to="us-gaap_FairValueAssetsMeasuredOnRecurringBasisUnobservableInputReconciliationByAssetClassDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_FairValueAssetsMeasuredOnRecurringBasisUnobservableInputReconciliationByAssetClassDomain" xlink:to="us-gaap_MoneyMarketFundsMember" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_FairValueAssetsMeasuredOnRecurringBasisUnobservableInputReconciliationByAssetClassDomain" xlink:to="us-gaap_USGovernmentAgenciesDebtSecuritiesMember" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisTable" xlink:to="us-gaap_FinancialInstrumentAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain" xlink:to="tlph_MarketableSecuritiesMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_FinancialInstrumentAxis" xlink:to="us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain-default" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisTable" xlink:to="us-gaap_FairValueByFairValueHierarchyLevelAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_FairValueMeasurementsFairValueHierarchyDomain" xlink:to="us-gaap_FairValueInputsLevel1Member" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_FairValueByFairValueHierarchyLevelAxis" xlink:to="us-gaap_FairValueMeasurementsFairValueHierarchyDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_FairValueMeasurementsFairValueHierarchyDomain" xlink:to="us-gaap_FairValueInputsLevel2Member" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_FairValueMeasurementsFairValueHierarchyDomain" xlink:to="us-gaap_FairValueInputsLevel3Member" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_FairValueAssetsMeasuredOnRecurringBasisUnobservableInputReconciliationByAssetClassDomain" xlink:to="tlph_WarrantLiabilityMember" xlink:type="arc"/>
    <link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisTable" xlink:to="us-gaap_ClassOfWarrantOrRightAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfWarrantOrRightDomain" xlink:to="tlph_CommonWarrantsMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_ClassOfWarrantOrRightAxis" xlink:to="us-gaap_ClassOfWarrantOrRightDomain-default" xlink:type="arc"/>
    <link:definitionArc order="5" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisTable" xlink:to="us-gaap_MeasurementInputTypeAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_MeasurementInputTypeDomain" xlink:to="us-gaap_MeasurementInputSharePriceMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_MeasurementInputTypeAxis" xlink:to="us-gaap_MeasurementInputTypeDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_MeasurementInputTypeDomain" xlink:to="us-gaap_MeasurementInputExpectedTermMember" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_MeasurementInputTypeDomain" xlink:to="us-gaap_MeasurementInputPriceVolatilityMember" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_MeasurementInputTypeDomain" xlink:to="us-gaap_MeasurementInputRiskFreeInterestRateMember" xlink:type="arc"/>
    <link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_MeasurementInputTypeDomain" xlink:to="us-gaap_MeasurementInputExpectedDividendRateMember" xlink:type="arc"/>
    <link:definitionArc order="6" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisTable" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DisclosureTextBlockAbstract" xlink:to="tlph_InvestmentsAndFairValueMeasurementDisclosureTextBlock" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.talphera.com/20260331/role/statement-note-3-discontinued-operations" xlink:href="tlph-20260331.xsd#statement-note-3-discontinued-operations" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://www.talphera.com/20260331/role/statement-note-3-discontinued-operations" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_MajorCustomersAxis" xlink:label="srt_MajorCustomersAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_NameOfMajorCustomerDomain" xlink:label="srt_NameOfMajorCustomerDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_NameOfMajorCustomerDomain" xlink:label="srt_NameOfMajorCustomerDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="tlph-20260331.xsd#tlph_DSUVIAMember" xlink:label="tlph_DSUVIAMember" xlink:type="locator"/>
    <link:loc xlink:href="tlph-20260331.xsd#tlph_DepartmentOfDefenseMember" xlink:label="tlph_DepartmentOfDefenseMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DisclosureTextBlockAbstract" xlink:label="us-gaap_DisclosureTextBlockAbstract" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DisposalGroupClassificationAxis" xlink:label="us-gaap_DisposalGroupClassificationAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DisposalGroupClassificationDomain" xlink:label="us-gaap_DisposalGroupClassificationDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DisposalGroupClassificationDomain" xlink:label="us-gaap_DisposalGroupClassificationDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DisposalGroupsIncludingDiscontinuedOperationsDisclosureTextBlock" xlink:label="us-gaap_DisposalGroupsIncludingDiscontinuedOperationsDisclosureTextBlock" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DisposalGroupsIncludingDiscontinuedOperationsNameDomain" xlink:label="us-gaap_DisposalGroupsIncludingDiscontinuedOperationsNameDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DisposalGroupsIncludingDiscontinuedOperationsNameDomain" xlink:label="us-gaap_DisposalGroupsIncludingDiscontinuedOperationsNameDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_IncomeStatementBalanceSheetAndAdditionalDisclosuresByDisposalGroupsIncludingDiscontinuedOperationsAxis" xlink:label="us-gaap_IncomeStatementBalanceSheetAndAdditionalDisclosuresByDisposalGroupsIncludingDiscontinuedOperationsAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_IncomeStatementBalanceSheetAndAdditionalDisclosuresByDisposalGroupsIncludingDiscontinuedOperationsTable" xlink:label="us-gaap_IncomeStatementBalanceSheetAndAdditionalDisclosuresByDisposalGroupsIncludingDiscontinuedOperationsTable" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SegmentDiscontinuedOperationsMember" xlink:label="us-gaap_SegmentDiscontinuedOperationsMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DisclosureTextBlockAbstract" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="srt_MajorCustomersAxis" xlink:to="srt_NameOfMajorCustomerDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_IncomeStatementBalanceSheetAndAdditionalDisclosuresByDisposalGroupsIncludingDiscontinuedOperationsAxis" xlink:to="us-gaap_DisposalGroupsIncludingDiscontinuedOperationsNameDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_DisposalGroupClassificationAxis" xlink:to="us-gaap_DisposalGroupClassificationDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_IncomeStatementBalanceSheetAndAdditionalDisclosuresByDisposalGroupsIncludingDiscontinuedOperationsTable" xlink:to="us-gaap_DisposalGroupClassificationAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DisposalGroupClassificationDomain" xlink:to="us-gaap_SegmentDiscontinuedOperationsMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_DisposalGroupClassificationAxis" xlink:to="us-gaap_DisposalGroupClassificationDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_IncomeStatementBalanceSheetAndAdditionalDisclosuresByDisposalGroupsIncludingDiscontinuedOperationsTable" xlink:to="us-gaap_IncomeStatementBalanceSheetAndAdditionalDisclosuresByDisposalGroupsIncludingDiscontinuedOperationsAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DisposalGroupsIncludingDiscontinuedOperationsNameDomain" xlink:to="tlph_DSUVIAMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_IncomeStatementBalanceSheetAndAdditionalDisclosuresByDisposalGroupsIncludingDiscontinuedOperationsAxis" xlink:to="us-gaap_DisposalGroupsIncludingDiscontinuedOperationsNameDomain-default" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_IncomeStatementBalanceSheetAndAdditionalDisclosuresByDisposalGroupsIncludingDiscontinuedOperationsTable" xlink:to="srt_MajorCustomersAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="srt_NameOfMajorCustomerDomain" xlink:to="tlph_DepartmentOfDefenseMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="srt_MajorCustomersAxis" xlink:to="srt_NameOfMajorCustomerDomain-default" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_IncomeStatementBalanceSheetAndAdditionalDisclosuresByDisposalGroupsIncludingDiscontinuedOperationsTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_IncomeStatementBalanceSheetAndAdditionalDisclosuresByDisposalGroupsIncludingDiscontinuedOperationsTable" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DisclosureTextBlockAbstract" xlink:to="us-gaap_DisposalGroupsIncludingDiscontinuedOperationsDisclosureTextBlock" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.talphera.com/20260331/role/statement-note-4-sale-of-future-payments" xlink:href="tlph-20260331.xsd#statement-note-4-sale-of-future-payments" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://www.talphera.com/20260331/role/statement-note-4-sale-of-future-payments" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_CounterpartyNameAxis" xlink:label="srt_CounterpartyNameAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_RepurchaseAgreementCounterpartyNameDomain" xlink:label="srt_RepurchaseAgreementCounterpartyNameDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_RepurchaseAgreementCounterpartyNameDomain" xlink:label="srt_RepurchaseAgreementCounterpartyNameDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="tlph-20260331.xsd#tlph_SaleOfFuturePaymentsTextBlock" xlink:label="tlph_SaleOfFuturePaymentsTextBlock" xlink:type="locator"/>
    <link:loc xlink:href="tlph-20260331.xsd#tlph_XOMAMember" xlink:label="tlph_XOMAMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DisclosureTextBlockAbstract" xlink:label="us-gaap_DisclosureTextBlockAbstract" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_IncomeStatementBalanceSheetAndAdditionalDisclosuresByDisposalGroupsIncludingDiscontinuedOperationsTable" xlink:label="us-gaap_IncomeStatementBalanceSheetAndAdditionalDisclosuresByDisposalGroupsIncludingDiscontinuedOperationsTable" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DisclosureTextBlockAbstract" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="srt_CounterpartyNameAxis" xlink:to="srt_RepurchaseAgreementCounterpartyNameDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_IncomeStatementBalanceSheetAndAdditionalDisclosuresByDisposalGroupsIncludingDiscontinuedOperationsTable" xlink:to="srt_CounterpartyNameAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="srt_RepurchaseAgreementCounterpartyNameDomain" xlink:to="tlph_XOMAMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="srt_CounterpartyNameAxis" xlink:to="srt_RepurchaseAgreementCounterpartyNameDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_IncomeStatementBalanceSheetAndAdditionalDisclosuresByDisposalGroupsIncludingDiscontinuedOperationsTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_IncomeStatementBalanceSheetAndAdditionalDisclosuresByDisposalGroupsIncludingDiscontinuedOperationsTable" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DisclosureTextBlockAbstract" xlink:to="tlph_SaleOfFuturePaymentsTextBlock" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.talphera.com/20260331/role/statement-note-6-stockholders-equity-" xlink:href="tlph-20260331.xsd#statement-note-6-stockholders-equity-" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://www.talphera.com/20260331/role/statement-note-6-stockholders-equity-" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="tlph-20260331.xsd#tlph_April2025PrivatePlacementThirdClosingMember" xlink:label="tlph_April2025PrivatePlacementThirdClosingMember" xlink:type="locator"/>
    <link:loc xlink:href="tlph-20260331.xsd#tlph_PrefundedWarrantsMember" xlink:label="tlph_PrefundedWarrantsMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfWarrantOrRightAxis" xlink:label="us-gaap_ClassOfWarrantOrRightAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfWarrantOrRightDomain" xlink:label="us-gaap_ClassOfWarrantOrRightDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfWarrantOrRightDomain" xlink:label="us-gaap_ClassOfWarrantOrRightDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DisclosureTextBlockAbstract" xlink:label="us-gaap_DisclosureTextBlockAbstract" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SaleOfStockNameOfTransactionDomain" xlink:label="us-gaap_SaleOfStockNameOfTransactionDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SaleOfStockNameOfTransactionDomain" xlink:label="us-gaap_SaleOfStockNameOfTransactionDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ScheduleOfStockByClassTable" xlink:label="us-gaap_ScheduleOfStockByClassTable" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StockholdersEquityNoteDisclosureTextBlock" xlink:label="us-gaap_StockholdersEquityNoteDisclosureTextBlock" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SubsidiarySaleOfStockAxis" xlink:label="us-gaap_SubsidiarySaleOfStockAxis" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DisclosureTextBlockAbstract" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_SubsidiarySaleOfStockAxis" xlink:to="us-gaap_SaleOfStockNameOfTransactionDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_ClassOfWarrantOrRightAxis" xlink:to="us-gaap_ClassOfWarrantOrRightDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_ScheduleOfStockByClassTable" xlink:to="us-gaap_SubsidiarySaleOfStockAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_SaleOfStockNameOfTransactionDomain" xlink:to="tlph_April2025PrivatePlacementThirdClosingMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_SubsidiarySaleOfStockAxis" xlink:to="us-gaap_SaleOfStockNameOfTransactionDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_ScheduleOfStockByClassTable" xlink:to="us-gaap_ClassOfWarrantOrRightAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfWarrantOrRightDomain" xlink:to="tlph_PrefundedWarrantsMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_ClassOfWarrantOrRightAxis" xlink:to="us-gaap_ClassOfWarrantOrRightDomain-default" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_ScheduleOfStockByClassTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ScheduleOfStockByClassTable" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DisclosureTextBlockAbstract" xlink:to="us-gaap_StockholdersEquityNoteDisclosureTextBlock" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.talphera.com/20260331/role/statement-note-7-warrants" xlink:href="tlph-20260331.xsd#statement-note-7-warrants" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://www.talphera.com/20260331/role/statement-note-7-warrants" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_RangeAxis" xlink:label="srt_RangeAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_RangeMember" xlink:label="srt_RangeMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_RangeMember" xlink:label="srt_RangeMember-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_WeightedAverageMember" xlink:label="srt_WeightedAverageMember" xlink:type="locator"/>
    <link:loc xlink:href="tlph-20260331.xsd#tlph_April2025PrefundedWarrantsMember" xlink:label="tlph_April2025PrefundedWarrantsMember" xlink:type="locator"/>
    <link:loc xlink:href="tlph-20260331.xsd#tlph_PrefundWarrantsMember" xlink:label="tlph_PrefundWarrantsMember" xlink:type="locator"/>
    <link:loc xlink:href="tlph-20260331.xsd#tlph_PrefundedWarrantsMember" xlink:label="tlph_PrefundedWarrantsMember" xlink:type="locator"/>
    <link:loc xlink:href="tlph-20260331.xsd#tlph_WarrantsDisclosureTextBlock" xlink:label="tlph_WarrantsDisclosureTextBlock" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfWarrantOrRightAxis" xlink:label="us-gaap_ClassOfWarrantOrRightAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfWarrantOrRightDomain" xlink:label="us-gaap_ClassOfWarrantOrRightDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfWarrantOrRightDomain" xlink:label="us-gaap_ClassOfWarrantOrRightDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfWarrantOrRightTable" xlink:label="us-gaap_ClassOfWarrantOrRightTable" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DisclosureTextBlockAbstract" xlink:label="us-gaap_DisclosureTextBlockAbstract" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DisclosureTextBlockAbstract" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="srt_RangeAxis" xlink:to="srt_RangeMember" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_ClassOfWarrantOrRightAxis" xlink:to="us-gaap_ClassOfWarrantOrRightDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_ClassOfWarrantOrRightTable" xlink:to="srt_RangeAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="srt_RangeMember" xlink:to="srt_WeightedAverageMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="srt_RangeAxis" xlink:to="srt_RangeMember-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_ClassOfWarrantOrRightTable" xlink:to="us-gaap_ClassOfWarrantOrRightAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfWarrantOrRightDomain" xlink:to="tlph_April2025PrefundedWarrantsMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_ClassOfWarrantOrRightAxis" xlink:to="us-gaap_ClassOfWarrantOrRightDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfWarrantOrRightDomain" xlink:to="tlph_PrefundedWarrantsMember" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfWarrantOrRightDomain" xlink:to="tlph_PrefundWarrantsMember" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_ClassOfWarrantOrRightTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ClassOfWarrantOrRightTable" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DisclosureTextBlockAbstract" xlink:to="tlph_WarrantsDisclosureTextBlock" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.talphera.com/20260331/role/statement-note-8-stockbased-compensation" xlink:href="tlph-20260331.xsd#statement-note-8-stockbased-compensation" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://www.talphera.com/20260331/role/statement-note-8-stockbased-compensation" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="tlph-20260331.xsd#tlph_AmendedEsppMember" xlink:label="tlph_AmendedEsppMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AwardTypeAxis" xlink:label="us-gaap_AwardTypeAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DisclosureOfCompensationRelatedCostsShareBasedPaymentsTextBlock" xlink:label="us-gaap_DisclosureOfCompensationRelatedCostsShareBasedPaymentsTextBlock" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DisclosureTextBlockAbstract" xlink:label="us-gaap_DisclosureTextBlockAbstract" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_PlanNameAxis" xlink:label="us-gaap_PlanNameAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_PlanNameDomain" xlink:label="us-gaap_PlanNameDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_PlanNameDomain" xlink:label="us-gaap_PlanNameDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_RestrictedStockUnitsRSUMember" xlink:label="us-gaap_RestrictedStockUnitsRSUMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ScheduleOfEmployeeServiceShareBasedCompensationAllocationOfRecognizedPeriodCostsTable" xlink:label="us-gaap_ScheduleOfEmployeeServiceShareBasedCompensationAllocationOfRecognizedPeriodCostsTable" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:label="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:label="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DisclosureTextBlockAbstract" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_PlanNameAxis" xlink:to="us-gaap_PlanNameDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_AwardTypeAxis" xlink:to="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_ScheduleOfEmployeeServiceShareBasedCompensationAllocationOfRecognizedPeriodCostsTable" xlink:to="us-gaap_AwardTypeAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:to="us-gaap_RestrictedStockUnitsRSUMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_AwardTypeAxis" xlink:to="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_ScheduleOfEmployeeServiceShareBasedCompensationAllocationOfRecognizedPeriodCostsTable" xlink:to="us-gaap_PlanNameAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_PlanNameDomain" xlink:to="tlph_AmendedEsppMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_PlanNameAxis" xlink:to="us-gaap_PlanNameDomain-default" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_ScheduleOfEmployeeServiceShareBasedCompensationAllocationOfRecognizedPeriodCostsTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ScheduleOfEmployeeServiceShareBasedCompensationAllocationOfRecognizedPeriodCostsTable" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DisclosureTextBlockAbstract" xlink:to="us-gaap_DisclosureOfCompensationRelatedCostsShareBasedPaymentsTextBlock" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.talphera.com/20260331/role/statement-note-9-net-loss-per-share-of-common-stock-" xlink:href="tlph-20260331.xsd#statement-note-9-net-loss-per-share-of-common-stock-" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://www.talphera.com/20260331/role/statement-note-9-net-loss-per-share-of-common-stock-" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="tlph-20260331.xsd#tlph_April2025PrefundedWarrantsMember" xlink:label="tlph_April2025PrefundedWarrantsMember" xlink:type="locator"/>
    <link:loc xlink:href="tlph-20260331.xsd#tlph_January2024PrefundedWarrantsMember" xlink:label="tlph_January2024PrefundedWarrantsMember" xlink:type="locator"/>
    <link:loc xlink:href="tlph-20260331.xsd#tlph_July2023PrefundedWarrantsMember" xlink:label="tlph_July2023PrefundedWarrantsMember" xlink:type="locator"/>
    <link:loc xlink:href="tlph-20260331.xsd#tlph_RSUsESPPAndEmployeeStockOptionsMember" xlink:label="tlph_RSUsESPPAndEmployeeStockOptionsMember" xlink:type="locator"/>
    <link:loc xlink:href="tlph-20260331.xsd#tlph_September2025PrefundedWarrantsMember" xlink:label="tlph_September2025PrefundedWarrantsMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareByAntidilutiveSecuritiesAxis" xlink:label="us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareByAntidilutiveSecuritiesAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AntidilutiveSecuritiesNameDomain" xlink:label="us-gaap_AntidilutiveSecuritiesNameDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AntidilutiveSecuritiesNameDomain" xlink:label="us-gaap_AntidilutiveSecuritiesNameDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfWarrantOrRightAxis" xlink:label="us-gaap_ClassOfWarrantOrRightAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfWarrantOrRightDomain" xlink:label="us-gaap_ClassOfWarrantOrRightDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfWarrantOrRightDomain" xlink:label="us-gaap_ClassOfWarrantOrRightDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DisclosureTextBlockAbstract" xlink:label="us-gaap_DisclosureTextBlockAbstract" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_EarningsPerShareTextBlock" xlink:label="us-gaap_EarningsPerShareTextBlock" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ScheduleOfEarningsPerShareBasicByCommonClassTable" xlink:label="us-gaap_ScheduleOfEarningsPerShareBasicByCommonClassTable" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_WarrantMember" xlink:label="us-gaap_WarrantMember" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DisclosureTextBlockAbstract" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_ClassOfWarrantOrRightAxis" xlink:to="us-gaap_ClassOfWarrantOrRightDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareByAntidilutiveSecuritiesAxis" xlink:to="us-gaap_AntidilutiveSecuritiesNameDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_ScheduleOfEarningsPerShareBasicByCommonClassTable" xlink:to="us-gaap_ClassOfWarrantOrRightAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfWarrantOrRightDomain" xlink:to="tlph_September2025PrefundedWarrantsMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_ClassOfWarrantOrRightAxis" xlink:to="us-gaap_ClassOfWarrantOrRightDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfWarrantOrRightDomain" xlink:to="tlph_April2025PrefundedWarrantsMember" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfWarrantOrRightDomain" xlink:to="tlph_January2024PrefundedWarrantsMember" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfWarrantOrRightDomain" xlink:to="tlph_July2023PrefundedWarrantsMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_ScheduleOfEarningsPerShareBasicByCommonClassTable" xlink:to="us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareByAntidilutiveSecuritiesAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AntidilutiveSecuritiesNameDomain" xlink:to="tlph_RSUsESPPAndEmployeeStockOptionsMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareByAntidilutiveSecuritiesAxis" xlink:to="us-gaap_AntidilutiveSecuritiesNameDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AntidilutiveSecuritiesNameDomain" xlink:to="us-gaap_WarrantMember" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_ScheduleOfEarningsPerShareBasicByCommonClassTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ScheduleOfEarningsPerShareBasicByCommonClassTable" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DisclosureTextBlockAbstract" xlink:to="us-gaap_EarningsPerShareTextBlock" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.talphera.com/20260331/role/statement-note-10-segment-information" xlink:href="tlph-20260331.xsd#statement-note-10-segment-information" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://www.talphera.com/20260331/role/statement-note-10-segment-information" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DisclosureTextBlockAbstract" xlink:label="us-gaap_DisclosureTextBlockAbstract" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ScheduleOfSegmentReportingInformationBySegmentTable" xlink:label="us-gaap_ScheduleOfSegmentReportingInformationBySegmentTable" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SegmentReportingDisclosureTextBlock" xlink:label="us-gaap_SegmentReportingDisclosureTextBlock" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DisclosureTextBlockAbstract" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_ScheduleOfSegmentReportingInformationBySegmentTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ScheduleOfSegmentReportingInformationBySegmentTable" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DisclosureTextBlockAbstract" xlink:to="us-gaap_SegmentReportingDisclosureTextBlock" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.talphera.com/20260331/role/statement-significant-accounting-policies-policies" xlink:href="tlph-20260331.xsd#statement-significant-accounting-policies-policies" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://www.talphera.com/20260331/role/statement-significant-accounting-policies-policies" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="tlph-20260331.xsd#tlph_LiquidityAndGoingConcernPolicyTextBlock" xlink:label="tlph_LiquidityAndGoingConcernPolicyTextBlock" xlink:type="locator"/>
    <link:loc xlink:href="tlph-20260331.xsd#tlph_PolicyTable" xlink:label="tlph_PolicyTable" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AccountingPoliciesAbstract" xlink:label="us-gaap_AccountingPoliciesAbstract" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_BasisOfAccountingPolicyPolicyTextBlock" xlink:label="us-gaap_BasisOfAccountingPolicyPolicyTextBlock" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_NewAccountingPronouncementsPolicyPolicyTextBlock" xlink:label="us-gaap_NewAccountingPronouncementsPolicyPolicyTextBlock" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_PolicyTextBlockAbstract" xlink:label="us-gaap_PolicyTextBlockAbstract" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_PolicyTextBlockAbstract" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="tlph_PolicyTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="tlph_PolicyTable" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_PolicyTextBlockAbstract" xlink:to="us-gaap_AccountingPoliciesAbstract" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AccountingPoliciesAbstract" xlink:to="tlph_LiquidityAndGoingConcernPolicyTextBlock" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AccountingPoliciesAbstract" xlink:to="us-gaap_BasisOfAccountingPolicyPolicyTextBlock" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AccountingPoliciesAbstract" xlink:to="us-gaap_NewAccountingPronouncementsPolicyPolicyTextBlock" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.talphera.com/20260331/role/statement-note-2-investments-and-fair-value-measurement-tables" xlink:href="tlph-20260331.xsd#statement-note-2-investments-and-fair-value-measurement-tables" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://www.talphera.com/20260331/role/statement-note-2-investments-and-fair-value-measurement-tables" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisTable" xlink:label="us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisTable" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisTableTextBlock" xlink:label="us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisTableTextBlock" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_FairValueLiabilitiesMeasuredOnRecurringBasisUnobservableInputReconciliationTextBlock" xlink:label="us-gaap_FairValueLiabilitiesMeasuredOnRecurringBasisUnobservableInputReconciliationTextBlock" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ScheduleOfCashCashEquivalentsAndShortTermInvestmentsTableTextBlock" xlink:label="us-gaap_ScheduleOfCashCashEquivalentsAndShortTermInvestmentsTableTextBlock" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_TableTextBlock" xlink:label="us-gaap_TableTextBlock" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_TableTextBlock" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisTable" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_TableTextBlock" xlink:to="us-gaap_ScheduleOfCashCashEquivalentsAndShortTermInvestmentsTableTextBlock" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_TableTextBlock" xlink:to="us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisTableTextBlock" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_TableTextBlock" xlink:to="us-gaap_FairValueLiabilitiesMeasuredOnRecurringBasisUnobservableInputReconciliationTextBlock" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.talphera.com/20260331/role/statement-note-3-discontinued-operations-tables" xlink:href="tlph-20260331.xsd#statement-note-3-discontinued-operations-tables" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://www.talphera.com/20260331/role/statement-note-3-discontinued-operations-tables" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_IncomeStatementBalanceSheetAndAdditionalDisclosuresByDisposalGroupsIncludingDiscontinuedOperationsTable" xlink:label="us-gaap_IncomeStatementBalanceSheetAndAdditionalDisclosuresByDisposalGroupsIncludingDiscontinuedOperationsTable" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ScheduleOfDisposalGroupsIncludingDiscontinuedOperationsIncomeStatementBalanceSheetAndAdditionalDisclosuresTextBlock" xlink:label="us-gaap_ScheduleOfDisposalGroupsIncludingDiscontinuedOperationsIncomeStatementBalanceSheetAndAdditionalDisclosuresTextBlock" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_TableTextBlock" xlink:label="us-gaap_TableTextBlock" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_TableTextBlock" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_IncomeStatementBalanceSheetAndAdditionalDisclosuresByDisposalGroupsIncludingDiscontinuedOperationsTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_IncomeStatementBalanceSheetAndAdditionalDisclosuresByDisposalGroupsIncludingDiscontinuedOperationsTable" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_TableTextBlock" xlink:to="us-gaap_ScheduleOfDisposalGroupsIncludingDiscontinuedOperationsIncomeStatementBalanceSheetAndAdditionalDisclosuresTextBlock" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.talphera.com/20260331/role/statement-note-7-warrants-tables" xlink:href="tlph-20260331.xsd#statement-note-7-warrants-tables" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://www.talphera.com/20260331/role/statement-note-7-warrants-tables" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfWarrantOrRightTable" xlink:label="us-gaap_ClassOfWarrantOrRightTable" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ScheduleOfStockholdersEquityNoteWarrantsOrRightsTextBlock" xlink:label="us-gaap_ScheduleOfStockholdersEquityNoteWarrantsOrRightsTextBlock" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_TableTextBlock" xlink:label="us-gaap_TableTextBlock" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_TableTextBlock" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_ClassOfWarrantOrRightTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ClassOfWarrantOrRightTable" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_TableTextBlock" xlink:to="us-gaap_ScheduleOfStockholdersEquityNoteWarrantsOrRightsTextBlock" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.talphera.com/20260331/role/statement-note-8-stockbased-compensation-tables" xlink:href="tlph-20260331.xsd#statement-note-8-stockbased-compensation-tables" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://www.talphera.com/20260331/role/statement-note-8-stockbased-compensation-tables" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ScheduleOfEmployeeServiceShareBasedCompensationAllocationOfRecognizedPeriodCostsTable" xlink:label="us-gaap_ScheduleOfEmployeeServiceShareBasedCompensationAllocationOfRecognizedPeriodCostsTable" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ScheduleOfEmployeeServiceShareBasedCompensationAllocationOfRecognizedPeriodCostsTextBlock" xlink:label="us-gaap_ScheduleOfEmployeeServiceShareBasedCompensationAllocationOfRecognizedPeriodCostsTextBlock" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ScheduleOfShareBasedCompensationStockOptionsActivityTableTextBlock" xlink:label="us-gaap_ScheduleOfShareBasedCompensationStockOptionsActivityTableTextBlock" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ScheduleOfSharebasedCompensationRestrictedStockAndRestrictedStockUnitsActivityTableTextBlock" xlink:label="us-gaap_ScheduleOfSharebasedCompensationRestrictedStockAndRestrictedStockUnitsActivityTableTextBlock" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_TableTextBlock" xlink:label="us-gaap_TableTextBlock" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_TableTextBlock" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_ScheduleOfEmployeeServiceShareBasedCompensationAllocationOfRecognizedPeriodCostsTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ScheduleOfEmployeeServiceShareBasedCompensationAllocationOfRecognizedPeriodCostsTable" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_TableTextBlock" xlink:to="us-gaap_ScheduleOfEmployeeServiceShareBasedCompensationAllocationOfRecognizedPeriodCostsTextBlock" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_TableTextBlock" xlink:to="us-gaap_ScheduleOfSharebasedCompensationRestrictedStockAndRestrictedStockUnitsActivityTableTextBlock" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_TableTextBlock" xlink:to="us-gaap_ScheduleOfShareBasedCompensationStockOptionsActivityTableTextBlock" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.talphera.com/20260331/role/statement-note-9-net-loss-per-share-of-common-stock-tables" xlink:href="tlph-20260331.xsd#statement-note-9-net-loss-per-share-of-common-stock-tables" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://www.talphera.com/20260331/role/statement-note-9-net-loss-per-share-of-common-stock-tables" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ScheduleOfAntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareTextBlock" xlink:label="us-gaap_ScheduleOfAntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareTextBlock" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ScheduleOfEarningsPerShareBasicByCommonClassTable" xlink:label="us-gaap_ScheduleOfEarningsPerShareBasicByCommonClassTable" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_TableTextBlock" xlink:label="us-gaap_TableTextBlock" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_TableTextBlock" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_ScheduleOfEarningsPerShareBasicByCommonClassTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ScheduleOfEarningsPerShareBasicByCommonClassTable" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_TableTextBlock" xlink:to="us-gaap_ScheduleOfAntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareTextBlock" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.talphera.com/20260331/role/statement-note-10-segment-information-tables" xlink:href="tlph-20260331.xsd#statement-note-10-segment-information-tables" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://www.talphera.com/20260331/role/statement-note-10-segment-information-tables" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ScheduleOfSegmentReportingInformationBySegmentTable" xlink:label="us-gaap_ScheduleOfSegmentReportingInformationBySegmentTable" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ScheduleOfSegmentReportingInformationBySegmentTextBlock" xlink:label="us-gaap_ScheduleOfSegmentReportingInformationBySegmentTextBlock" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_TableTextBlock" xlink:label="us-gaap_TableTextBlock" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_TableTextBlock" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_ScheduleOfSegmentReportingInformationBySegmentTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ScheduleOfSegmentReportingInformationBySegmentTable" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_TableTextBlock" xlink:to="us-gaap_ScheduleOfSegmentReportingInformationBySegmentTextBlock" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.talphera.com/20260331/role/statement-note-2-investments-and-fair-value-measurement-details-textual" xlink:href="tlph-20260331.xsd#statement-note-2-investments-and-fair-value-measurement-details-textual" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://www.talphera.com/20260331/role/statement-note-2-investments-and-fair-value-measurement-details-textual" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="tlph-20260331.xsd#tlph_CommonWarrantsMember" xlink:label="tlph_CommonWarrantsMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfWarrantOrRightAxis" xlink:label="us-gaap_ClassOfWarrantOrRightAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfWarrantOrRightDomain" xlink:label="us-gaap_ClassOfWarrantOrRightDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfWarrantOrRightDomain" xlink:label="us-gaap_ClassOfWarrantOrRightDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfWarrantOrRightExercisePriceOfWarrantsOrRights1" xlink:label="us-gaap_ClassOfWarrantOrRightExercisePriceOfWarrantsOrRights1" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DebtSecuritiesAvailableForSaleAllowanceForCreditLoss" xlink:label="us-gaap_DebtSecuritiesAvailableForSaleAllowanceForCreditLoss" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisTable" xlink:label="us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisTable" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_MarketableSecuritiesGainLossExcludingOtherThanTemporaryImpairments" xlink:label="us-gaap_MarketableSecuritiesGainLossExcludingOtherThanTemporaryImpairments" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_MeasurementInputExpectedDividendRateMember" xlink:label="us-gaap_MeasurementInputExpectedDividendRateMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_MeasurementInputExpectedTermMember" xlink:label="us-gaap_MeasurementInputExpectedTermMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_MeasurementInputPriceVolatilityMember" xlink:label="us-gaap_MeasurementInputPriceVolatilityMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_MeasurementInputRiskFreeInterestRateMember" xlink:label="us-gaap_MeasurementInputRiskFreeInterestRateMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_MeasurementInputSharePriceMember" xlink:label="us-gaap_MeasurementInputSharePriceMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_MeasurementInputTypeAxis" xlink:label="us-gaap_MeasurementInputTypeAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_MeasurementInputTypeDomain" xlink:label="us-gaap_MeasurementInputTypeDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_MeasurementInputTypeDomain" xlink:label="us-gaap_MeasurementInputTypeDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_WarrantsAndRightsOutstanding" xlink:label="us-gaap_WarrantsAndRightsOutstanding" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_WarrantsAndRightsOutstandingMeasurementInput" xlink:label="us-gaap_WarrantsAndRightsOutstandingMeasurementInput" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_ClassOfWarrantOrRightAxis" xlink:to="us-gaap_ClassOfWarrantOrRightDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_MeasurementInputTypeAxis" xlink:to="us-gaap_MeasurementInputTypeDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisTable" xlink:to="us-gaap_ClassOfWarrantOrRightAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfWarrantOrRightDomain" xlink:to="tlph_CommonWarrantsMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_ClassOfWarrantOrRightAxis" xlink:to="us-gaap_ClassOfWarrantOrRightDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisTable" xlink:to="us-gaap_MeasurementInputTypeAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_MeasurementInputTypeDomain" xlink:to="us-gaap_MeasurementInputSharePriceMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_MeasurementInputTypeAxis" xlink:to="us-gaap_MeasurementInputTypeDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_MeasurementInputTypeDomain" xlink:to="us-gaap_MeasurementInputExpectedTermMember" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_MeasurementInputTypeDomain" xlink:to="us-gaap_MeasurementInputPriceVolatilityMember" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_MeasurementInputTypeDomain" xlink:to="us-gaap_MeasurementInputRiskFreeInterestRateMember" xlink:type="arc"/>
    <link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_MeasurementInputTypeDomain" xlink:to="us-gaap_MeasurementInputExpectedDividendRateMember" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisTable" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_MarketableSecuritiesGainLossExcludingOtherThanTemporaryImpairments" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DebtSecuritiesAvailableForSaleAllowanceForCreditLoss" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_WarrantsAndRightsOutstanding" xlink:type="arc"/>
    <link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ClassOfWarrantOrRightExercisePriceOfWarrantsOrRights1" xlink:type="arc"/>
    <link:definitionArc order="5" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_WarrantsAndRightsOutstandingMeasurementInput" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.talphera.com/20260331/role/statement-note-2-investments-and-fair-value-measurement-summary-of-cash-cash-equivalents-and-investments-details" xlink:href="tlph-20260331.xsd#statement-note-2-investments-and-fair-value-measurement-summary-of-cash-cash-equivalents-and-investments-details" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://www.talphera.com/20260331/role/statement-note-2-investments-and-fair-value-measurement-summary-of-cash-cash-equivalents-and-investments-details" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="tlph-20260331.xsd#tlph_CashCashEquivalentsAndShorttermInvestmentsFairValue" xlink:label="tlph_CashCashEquivalentsAndShorttermInvestmentsFairValue" xlink:type="locator"/>
    <link:loc xlink:href="tlph-20260331.xsd#tlph_MarketableSecuritiesMember" xlink:label="tlph_MarketableSecuritiesMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AvailableForSaleDebtSecuritiesAccumulatedGrossUnrealizedGainBeforeTax" xlink:label="us-gaap_AvailableForSaleDebtSecuritiesAccumulatedGrossUnrealizedGainBeforeTax" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AvailableForSaleDebtSecuritiesAmortizedCostBasis" xlink:label="us-gaap_AvailableForSaleDebtSecuritiesAmortizedCostBasis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AvailableForSaleSecuritiesDebtSecurities" xlink:label="us-gaap_AvailableForSaleSecuritiesDebtSecurities" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_Cash" xlink:label="us-gaap_Cash" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_CashAndCashEquivalentsAxis" xlink:label="us-gaap_CashAndCashEquivalentsAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_CashAndCashEquivalentsFairValueDisclosure" xlink:label="us-gaap_CashAndCashEquivalentsFairValueDisclosure" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_CashAndCashEquivalentsMember" xlink:label="us-gaap_CashAndCashEquivalentsMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_CashCashEquivalentsAndShortTermInvestments" xlink:label="us-gaap_CashCashEquivalentsAndShortTermInvestments" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_CashCashEquivalentsRestrictedCashAndRestrictedCashEquivalentsIncludingDisposalGroupAndDiscontinuedOperations" xlink:label="us-gaap_CashCashEquivalentsRestrictedCashAndRestrictedCashEquivalentsIncludingDisposalGroupAndDiscontinuedOperations" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_CashMember" xlink:label="us-gaap_CashMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_FairValueAssetsMeasuredOnRecurringBasisUnobservableInputReconciliationByAssetClassDomain" xlink:label="us-gaap_FairValueAssetsMeasuredOnRecurringBasisUnobservableInputReconciliationByAssetClassDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_FairValueAssetsMeasuredOnRecurringBasisUnobservableInputReconciliationByAssetClassDomain" xlink:label="us-gaap_FairValueAssetsMeasuredOnRecurringBasisUnobservableInputReconciliationByAssetClassDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_FairValueByAssetClassAxis" xlink:label="us-gaap_FairValueByAssetClassAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_FinancialInstrumentAxis" xlink:label="us-gaap_FinancialInstrumentAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_MoneyMarketFundsAtCarryingValue" xlink:label="us-gaap_MoneyMarketFundsAtCarryingValue" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_MoneyMarketFundsMember" xlink:label="us-gaap_MoneyMarketFundsMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_RestrictedCashAndCashEquivalentsCashAndCashEquivalentsMember" xlink:label="us-gaap_RestrictedCashAndCashEquivalentsCashAndCashEquivalentsMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_RestrictedCashAndCashEquivalentsCashAndCashEquivalentsMember" xlink:label="us-gaap_RestrictedCashAndCashEquivalentsCashAndCashEquivalentsMember-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ScheduleOfCashAndCashEquivalentsTable" xlink:label="us-gaap_ScheduleOfCashAndCashEquivalentsTable" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain" xlink:label="us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain" xlink:label="us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_USGovernmentAgenciesDebtSecuritiesMember" xlink:label="us-gaap_USGovernmentAgenciesDebtSecuritiesMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_USGovernmentAgenciesSecuritiesAtCarryingValue" xlink:label="us-gaap_USGovernmentAgenciesSecuritiesAtCarryingValue" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_FairValueByAssetClassAxis" xlink:to="us-gaap_FairValueAssetsMeasuredOnRecurringBasisUnobservableInputReconciliationByAssetClassDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_FinancialInstrumentAxis" xlink:to="us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_CashAndCashEquivalentsAxis" xlink:to="us-gaap_RestrictedCashAndCashEquivalentsCashAndCashEquivalentsMember" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_ScheduleOfCashAndCashEquivalentsTable" xlink:to="us-gaap_CashAndCashEquivalentsAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_RestrictedCashAndCashEquivalentsCashAndCashEquivalentsMember" xlink:to="us-gaap_CashAndCashEquivalentsMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_CashAndCashEquivalentsAxis" xlink:to="us-gaap_RestrictedCashAndCashEquivalentsCashAndCashEquivalentsMember-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_ScheduleOfCashAndCashEquivalentsTable" xlink:to="us-gaap_FairValueByAssetClassAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_FairValueAssetsMeasuredOnRecurringBasisUnobservableInputReconciliationByAssetClassDomain" xlink:to="us-gaap_CashMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_FairValueByAssetClassAxis" xlink:to="us-gaap_FairValueAssetsMeasuredOnRecurringBasisUnobservableInputReconciliationByAssetClassDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_FairValueAssetsMeasuredOnRecurringBasisUnobservableInputReconciliationByAssetClassDomain" xlink:to="us-gaap_MoneyMarketFundsMember" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_FairValueAssetsMeasuredOnRecurringBasisUnobservableInputReconciliationByAssetClassDomain" xlink:to="us-gaap_USGovernmentAgenciesDebtSecuritiesMember" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_ScheduleOfCashAndCashEquivalentsTable" xlink:to="us-gaap_FinancialInstrumentAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain" xlink:to="tlph_MarketableSecuritiesMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_FinancialInstrumentAxis" xlink:to="us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain-default" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_ScheduleOfCashAndCashEquivalentsTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ScheduleOfCashAndCashEquivalentsTable" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_Cash" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_CashAndCashEquivalentsFairValueDisclosure" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_MoneyMarketFundsAtCarryingValue" xlink:type="arc"/>
    <link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_USGovernmentAgenciesSecuritiesAtCarryingValue" xlink:type="arc"/>
    <link:definitionArc order="5" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_CashCashEquivalentsRestrictedCashAndRestrictedCashEquivalentsIncludingDisposalGroupAndDiscontinuedOperations" xlink:type="arc"/>
    <link:definitionArc order="6" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_AvailableForSaleDebtSecuritiesAmortizedCostBasis" xlink:type="arc"/>
    <link:definitionArc order="7" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_AvailableForSaleSecuritiesDebtSecurities" xlink:type="arc"/>
    <link:definitionArc order="8" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_AvailableForSaleDebtSecuritiesAccumulatedGrossUnrealizedGainBeforeTax" xlink:type="arc"/>
    <link:definitionArc order="9" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_CashCashEquivalentsAndShortTermInvestments" xlink:type="arc"/>
    <link:definitionArc order="10" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="tlph_CashCashEquivalentsAndShorttermInvestmentsFairValue" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.talphera.com/20260331/role/statement-note-2-investments-and-fair-value-measurement-fair-value-of-financial-assets-and-liabilities-by-level-within-fair-value-hierarchy-details" xlink:href="tlph-20260331.xsd#statement-note-2-investments-and-fair-value-measurement-fair-value-of-financial-assets-and-liabilities-by-level-within-fair-value-hierarchy-details" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://www.talphera.com/20260331/role/statement-note-2-investments-and-fair-value-measurement-fair-value-of-financial-assets-and-liabilities-by-level-within-fair-value-hierarchy-details" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="tlph-20260331.xsd#tlph_WarrantLiabilityMember" xlink:label="tlph_WarrantLiabilityMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AssetsFairValueDisclosure" xlink:label="us-gaap_AssetsFairValueDisclosure" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisTable" xlink:label="us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisTable" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_FairValueAssetsMeasuredOnRecurringBasisUnobservableInputReconciliationByAssetClassDomain" xlink:label="us-gaap_FairValueAssetsMeasuredOnRecurringBasisUnobservableInputReconciliationByAssetClassDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_FairValueAssetsMeasuredOnRecurringBasisUnobservableInputReconciliationByAssetClassDomain" xlink:label="us-gaap_FairValueAssetsMeasuredOnRecurringBasisUnobservableInputReconciliationByAssetClassDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_FairValueByAssetClassAxis" xlink:label="us-gaap_FairValueByAssetClassAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_FairValueByFairValueHierarchyLevelAxis" xlink:label="us-gaap_FairValueByFairValueHierarchyLevelAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_FairValueInputsLevel1Member" xlink:label="us-gaap_FairValueInputsLevel1Member" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_FairValueInputsLevel2Member" xlink:label="us-gaap_FairValueInputsLevel2Member" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_FairValueInputsLevel3Member" xlink:label="us-gaap_FairValueInputsLevel3Member" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_FairValueMeasurementsFairValueHierarchyDomain" xlink:label="us-gaap_FairValueMeasurementsFairValueHierarchyDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_FairValueMeasurementsFairValueHierarchyDomain" xlink:label="us-gaap_FairValueMeasurementsFairValueHierarchyDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_LiabilitiesFairValueDisclosure" xlink:label="us-gaap_LiabilitiesFairValueDisclosure" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_MoneyMarketFundsMember" xlink:label="us-gaap_MoneyMarketFundsMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_USGovernmentAgenciesDebtSecuritiesMember" xlink:label="us-gaap_USGovernmentAgenciesDebtSecuritiesMember" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_FairValueByAssetClassAxis" xlink:to="us-gaap_FairValueAssetsMeasuredOnRecurringBasisUnobservableInputReconciliationByAssetClassDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_FairValueByFairValueHierarchyLevelAxis" xlink:to="us-gaap_FairValueMeasurementsFairValueHierarchyDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisTable" xlink:to="us-gaap_FairValueByAssetClassAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_FairValueAssetsMeasuredOnRecurringBasisUnobservableInputReconciliationByAssetClassDomain" xlink:to="us-gaap_MoneyMarketFundsMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_FairValueByAssetClassAxis" xlink:to="us-gaap_FairValueAssetsMeasuredOnRecurringBasisUnobservableInputReconciliationByAssetClassDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisTable" xlink:to="us-gaap_FairValueByFairValueHierarchyLevelAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_FairValueMeasurementsFairValueHierarchyDomain" xlink:to="us-gaap_FairValueInputsLevel1Member" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_FairValueByFairValueHierarchyLevelAxis" xlink:to="us-gaap_FairValueMeasurementsFairValueHierarchyDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_FairValueMeasurementsFairValueHierarchyDomain" xlink:to="us-gaap_FairValueInputsLevel2Member" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_FairValueMeasurementsFairValueHierarchyDomain" xlink:to="us-gaap_FairValueInputsLevel3Member" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_FairValueAssetsMeasuredOnRecurringBasisUnobservableInputReconciliationByAssetClassDomain" xlink:to="us-gaap_USGovernmentAgenciesDebtSecuritiesMember" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_FairValueAssetsMeasuredOnRecurringBasisUnobservableInputReconciliationByAssetClassDomain" xlink:to="tlph_WarrantLiabilityMember" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisTable" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_AssetsFairValueDisclosure" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_LiabilitiesFairValueDisclosure" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.talphera.com/20260331/role/statement-note-2-investments-and-fair-value-measurement-summary-of-changes-in-fair-value-of-level-iii-financial-liabilities-details" xlink:href="tlph-20260331.xsd#statement-note-2-investments-and-fair-value-measurement-summary-of-changes-in-fair-value-of-level-iii-financial-liabilities-details" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://www.talphera.com/20260331/role/statement-note-2-investments-and-fair-value-measurement-summary-of-changes-in-fair-value-of-level-iii-financial-liabilities-details" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisTable" xlink:label="us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisTable" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_FairValueByFairValueHierarchyLevelAxis" xlink:label="us-gaap_FairValueByFairValueHierarchyLevelAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_FairValueInputsLevel3Member" xlink:label="us-gaap_FairValueInputsLevel3Member" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_FairValueMeasurementWithUnobservableInputsReconciliationRecurringBasisLiabilityGainLossIncludedInEarnings" xlink:label="us-gaap_FairValueMeasurementWithUnobservableInputsReconciliationRecurringBasisLiabilityGainLossIncludedInEarnings" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_FairValueMeasurementWithUnobservableInputsReconciliationsRecurringBasisLiabilityValue" xlink:label="us-gaap_FairValueMeasurementWithUnobservableInputsReconciliationsRecurringBasisLiabilityValue" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_FairValueMeasurementsFairValueHierarchyDomain" xlink:label="us-gaap_FairValueMeasurementsFairValueHierarchyDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_FairValueMeasurementsFairValueHierarchyDomain" xlink:label="us-gaap_FairValueMeasurementsFairValueHierarchyDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_FairValueByFairValueHierarchyLevelAxis" xlink:to="us-gaap_FairValueMeasurementsFairValueHierarchyDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisTable" xlink:to="us-gaap_FairValueByFairValueHierarchyLevelAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_FairValueMeasurementsFairValueHierarchyDomain" xlink:to="us-gaap_FairValueInputsLevel3Member" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_FairValueByFairValueHierarchyLevelAxis" xlink:to="us-gaap_FairValueMeasurementsFairValueHierarchyDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisTable" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_FairValueMeasurementWithUnobservableInputsReconciliationsRecurringBasisLiabilityValue" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_FairValueMeasurementWithUnobservableInputsReconciliationRecurringBasisLiabilityGainLossIncludedInEarnings" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.talphera.com/20260331/role/statement-note-3-discontinued-operations-details-textual" xlink:href="tlph-20260331.xsd#statement-note-3-discontinued-operations-details-textual" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://www.talphera.com/20260331/role/statement-note-3-discontinued-operations-details-textual" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_MajorCustomersAxis" xlink:label="srt_MajorCustomersAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_NameOfMajorCustomerDomain" xlink:label="srt_NameOfMajorCustomerDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_NameOfMajorCustomerDomain" xlink:label="srt_NameOfMajorCustomerDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="tlph-20260331.xsd#tlph_DSUVIAMember" xlink:label="tlph_DSUVIAMember" xlink:type="locator"/>
    <link:loc xlink:href="tlph-20260331.xsd#tlph_DepartmentOfDefenseMember" xlink:label="tlph_DepartmentOfDefenseMember" xlink:type="locator"/>
    <link:loc xlink:href="tlph-20260331.xsd#tlph_DisposalGroupIncludingDiscontinuedOperationMaximumAmountInSalesbasedMilestone" xlink:label="tlph_DisposalGroupIncludingDiscontinuedOperationMaximumAmountInSalesbasedMilestone" xlink:type="locator"/>
    <link:loc xlink:href="tlph-20260331.xsd#tlph_DisposalGroupIncludingDiscontinuedOperationPercentageOfRoyaltyOnCommercialSales" xlink:label="tlph_DisposalGroupIncludingDiscontinuedOperationPercentageOfRoyaltyOnCommercialSales" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DisposalGroupClassificationAxis" xlink:label="us-gaap_DisposalGroupClassificationAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DisposalGroupClassificationDomain" xlink:label="us-gaap_DisposalGroupClassificationDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DisposalGroupClassificationDomain" xlink:label="us-gaap_DisposalGroupClassificationDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DisposalGroupIncludingDiscontinuedOperationConsideration" xlink:label="us-gaap_DisposalGroupIncludingDiscontinuedOperationConsideration" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DisposalGroupsIncludingDiscontinuedOperationsNameDomain" xlink:label="us-gaap_DisposalGroupsIncludingDiscontinuedOperationsNameDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DisposalGroupsIncludingDiscontinuedOperationsNameDomain" xlink:label="us-gaap_DisposalGroupsIncludingDiscontinuedOperationsNameDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_IncomeStatementBalanceSheetAndAdditionalDisclosuresByDisposalGroupsIncludingDiscontinuedOperationsAxis" xlink:label="us-gaap_IncomeStatementBalanceSheetAndAdditionalDisclosuresByDisposalGroupsIncludingDiscontinuedOperationsAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_IncomeStatementBalanceSheetAndAdditionalDisclosuresByDisposalGroupsIncludingDiscontinuedOperationsTable" xlink:label="us-gaap_IncomeStatementBalanceSheetAndAdditionalDisclosuresByDisposalGroupsIncludingDiscontinuedOperationsTable" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SegmentDiscontinuedOperationsMember" xlink:label="us-gaap_SegmentDiscontinuedOperationsMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="srt_MajorCustomersAxis" xlink:to="srt_NameOfMajorCustomerDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_IncomeStatementBalanceSheetAndAdditionalDisclosuresByDisposalGroupsIncludingDiscontinuedOperationsAxis" xlink:to="us-gaap_DisposalGroupsIncludingDiscontinuedOperationsNameDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_DisposalGroupClassificationAxis" xlink:to="us-gaap_DisposalGroupClassificationDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_IncomeStatementBalanceSheetAndAdditionalDisclosuresByDisposalGroupsIncludingDiscontinuedOperationsTable" xlink:to="us-gaap_DisposalGroupClassificationAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DisposalGroupClassificationDomain" xlink:to="us-gaap_SegmentDiscontinuedOperationsMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_DisposalGroupClassificationAxis" xlink:to="us-gaap_DisposalGroupClassificationDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_IncomeStatementBalanceSheetAndAdditionalDisclosuresByDisposalGroupsIncludingDiscontinuedOperationsTable" xlink:to="us-gaap_IncomeStatementBalanceSheetAndAdditionalDisclosuresByDisposalGroupsIncludingDiscontinuedOperationsAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DisposalGroupsIncludingDiscontinuedOperationsNameDomain" xlink:to="tlph_DSUVIAMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_IncomeStatementBalanceSheetAndAdditionalDisclosuresByDisposalGroupsIncludingDiscontinuedOperationsAxis" xlink:to="us-gaap_DisposalGroupsIncludingDiscontinuedOperationsNameDomain-default" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_IncomeStatementBalanceSheetAndAdditionalDisclosuresByDisposalGroupsIncludingDiscontinuedOperationsTable" xlink:to="srt_MajorCustomersAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="srt_NameOfMajorCustomerDomain" xlink:to="tlph_DepartmentOfDefenseMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="srt_MajorCustomersAxis" xlink:to="srt_NameOfMajorCustomerDomain-default" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_IncomeStatementBalanceSheetAndAdditionalDisclosuresByDisposalGroupsIncludingDiscontinuedOperationsTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_IncomeStatementBalanceSheetAndAdditionalDisclosuresByDisposalGroupsIncludingDiscontinuedOperationsTable" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DisposalGroupIncludingDiscontinuedOperationConsideration" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="tlph_DisposalGroupIncludingDiscontinuedOperationPercentageOfRoyaltyOnCommercialSales" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="tlph_DisposalGroupIncludingDiscontinuedOperationMaximumAmountInSalesbasedMilestone" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.talphera.com/20260331/role/statement-note-3-discontinued-operations-summary-of-discontinued-operation-details" xlink:href="tlph-20260331.xsd#statement-note-3-discontinued-operations-summary-of-discontinued-operation-details" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://www.talphera.com/20260331/role/statement-note-3-discontinued-operations-summary-of-discontinued-operation-details" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="tlph-20260331.xsd#tlph_DSUVIAMember" xlink:label="tlph_DSUVIAMember" xlink:type="locator"/>
    <link:loc xlink:href="tlph-20260331.xsd#tlph_DiscontinuedOperationChangeInEstimatedNetLiabilities" xlink:label="tlph_DiscontinuedOperationChangeInEstimatedNetLiabilities" xlink:type="locator"/>
    <link:loc xlink:href="tlph-20260331.xsd#tlph_DisposalGroupIncludingDiscontinuedOperationNetAssets" xlink:label="tlph_DisposalGroupIncludingDiscontinuedOperationNetAssets" xlink:type="locator"/>
    <link:loc xlink:href="tlph-20260331.xsd#tlph_IncomeLossFromDiscontinuedOperationsNetOfTaxAttributableToParentBeforeInterest" xlink:label="tlph_IncomeLossFromDiscontinuedOperationsNetOfTaxAttributableToParentBeforeInterest" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DisposalGroupClassificationAxis" xlink:label="us-gaap_DisposalGroupClassificationAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DisposalGroupClassificationDomain" xlink:label="us-gaap_DisposalGroupClassificationDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DisposalGroupClassificationDomain" xlink:label="us-gaap_DisposalGroupClassificationDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DisposalGroupIncludingDiscontinuedOperationAccruedLiabilitiesCurrent" xlink:label="us-gaap_DisposalGroupIncludingDiscontinuedOperationAccruedLiabilitiesCurrent" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DisposalGroupsIncludingDiscontinuedOperationsNameDomain" xlink:label="us-gaap_DisposalGroupsIncludingDiscontinuedOperationsNameDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DisposalGroupsIncludingDiscontinuedOperationsNameDomain" xlink:label="us-gaap_DisposalGroupsIncludingDiscontinuedOperationsNameDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_IncomeStatementBalanceSheetAndAdditionalDisclosuresByDisposalGroupsIncludingDiscontinuedOperationsAxis" xlink:label="us-gaap_IncomeStatementBalanceSheetAndAdditionalDisclosuresByDisposalGroupsIncludingDiscontinuedOperationsAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_IncomeStatementBalanceSheetAndAdditionalDisclosuresByDisposalGroupsIncludingDiscontinuedOperationsTable" xlink:label="us-gaap_IncomeStatementBalanceSheetAndAdditionalDisclosuresByDisposalGroupsIncludingDiscontinuedOperationsTable" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_LiabilitiesOfDisposalGroupIncludingDiscontinuedOperationCurrent" xlink:label="us-gaap_LiabilitiesOfDisposalGroupIncludingDiscontinuedOperationCurrent" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SegmentDiscontinuedOperationsMember" xlink:label="us-gaap_SegmentDiscontinuedOperationsMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_IncomeStatementBalanceSheetAndAdditionalDisclosuresByDisposalGroupsIncludingDiscontinuedOperationsAxis" xlink:to="us-gaap_DisposalGroupsIncludingDiscontinuedOperationsNameDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_DisposalGroupClassificationAxis" xlink:to="us-gaap_DisposalGroupClassificationDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_IncomeStatementBalanceSheetAndAdditionalDisclosuresByDisposalGroupsIncludingDiscontinuedOperationsTable" xlink:to="us-gaap_DisposalGroupClassificationAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DisposalGroupClassificationDomain" xlink:to="us-gaap_SegmentDiscontinuedOperationsMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_DisposalGroupClassificationAxis" xlink:to="us-gaap_DisposalGroupClassificationDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_IncomeStatementBalanceSheetAndAdditionalDisclosuresByDisposalGroupsIncludingDiscontinuedOperationsTable" xlink:to="us-gaap_IncomeStatementBalanceSheetAndAdditionalDisclosuresByDisposalGroupsIncludingDiscontinuedOperationsAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DisposalGroupsIncludingDiscontinuedOperationsNameDomain" xlink:to="tlph_DSUVIAMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_IncomeStatementBalanceSheetAndAdditionalDisclosuresByDisposalGroupsIncludingDiscontinuedOperationsAxis" xlink:to="us-gaap_DisposalGroupsIncludingDiscontinuedOperationsNameDomain-default" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_IncomeStatementBalanceSheetAndAdditionalDisclosuresByDisposalGroupsIncludingDiscontinuedOperationsTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_IncomeStatementBalanceSheetAndAdditionalDisclosuresByDisposalGroupsIncludingDiscontinuedOperationsTable" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="tlph_DiscontinuedOperationChangeInEstimatedNetLiabilities" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DisposalGroupIncludingDiscontinuedOperationAccruedLiabilitiesCurrent" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="tlph_IncomeLossFromDiscontinuedOperationsNetOfTaxAttributableToParentBeforeInterest" xlink:type="arc"/>
    <link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_LiabilitiesOfDisposalGroupIncludingDiscontinuedOperationCurrent" xlink:type="arc"/>
    <link:definitionArc order="5" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="tlph_DisposalGroupIncludingDiscontinuedOperationNetAssets" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.talphera.com/20260331/role/statement-note-4-sale-of-future-payments-details-textual" xlink:href="tlph-20260331.xsd#statement-note-4-sale-of-future-payments-details-textual" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://www.talphera.com/20260331/role/statement-note-4-sale-of-future-payments-details-textual" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_CounterpartyNameAxis" xlink:label="srt_CounterpartyNameAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_RepurchaseAgreementCounterpartyNameDomain" xlink:label="srt_RepurchaseAgreementCounterpartyNameDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_RepurchaseAgreementCounterpartyNameDomain" xlink:label="srt_RepurchaseAgreementCounterpartyNameDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="tlph-20260331.xsd#tlph_ProceedsFromSaleOfReceivables" xlink:label="tlph_ProceedsFromSaleOfReceivables" xlink:type="locator"/>
    <link:loc xlink:href="tlph-20260331.xsd#tlph_XOMAMember" xlink:label="tlph_XOMAMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_IncomeStatementBalanceSheetAndAdditionalDisclosuresByDisposalGroupsIncludingDiscontinuedOperationsTable" xlink:label="us-gaap_IncomeStatementBalanceSheetAndAdditionalDisclosuresByDisposalGroupsIncludingDiscontinuedOperationsTable" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ProceedsFromDebtNetOfIssuanceCosts" xlink:label="us-gaap_ProceedsFromDebtNetOfIssuanceCosts" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="srt_CounterpartyNameAxis" xlink:to="srt_RepurchaseAgreementCounterpartyNameDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_IncomeStatementBalanceSheetAndAdditionalDisclosuresByDisposalGroupsIncludingDiscontinuedOperationsTable" xlink:to="srt_CounterpartyNameAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="srt_RepurchaseAgreementCounterpartyNameDomain" xlink:to="tlph_XOMAMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="srt_CounterpartyNameAxis" xlink:to="srt_RepurchaseAgreementCounterpartyNameDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_IncomeStatementBalanceSheetAndAdditionalDisclosuresByDisposalGroupsIncludingDiscontinuedOperationsTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_IncomeStatementBalanceSheetAndAdditionalDisclosuresByDisposalGroupsIncludingDiscontinuedOperationsTable" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="tlph_ProceedsFromSaleOfReceivables" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ProceedsFromDebtNetOfIssuanceCosts" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.talphera.com/20260331/role/statement-note-6-stockholders-equity-details-textual" xlink:href="tlph-20260331.xsd#statement-note-6-stockholders-equity-details-textual" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://www.talphera.com/20260331/role/statement-note-6-stockholders-equity-details-textual" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="tlph-20260331.xsd#tlph_April2025PrivatePlacementThirdClosingMember" xlink:label="tlph_April2025PrivatePlacementThirdClosingMember" xlink:type="locator"/>
    <link:loc xlink:href="tlph-20260331.xsd#tlph_ClassOfWarrantOrRightIssuedDuringPeriodPricePerWarrant" xlink:label="tlph_ClassOfWarrantOrRightIssuedDuringPeriodPricePerWarrant" xlink:type="locator"/>
    <link:loc xlink:href="tlph-20260331.xsd#tlph_PrefundedWarrantsMember" xlink:label="tlph_PrefundedWarrantsMember" xlink:type="locator"/>
    <link:loc xlink:href="tlph-20260331.xsd#tlph_PrivatePlacementCovenantToTriggerSalePatientsEnrolled" xlink:label="tlph_PrivatePlacementCovenantToTriggerSalePatientsEnrolled" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfWarrantOrRightAxis" xlink:label="us-gaap_ClassOfWarrantOrRightAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfWarrantOrRightDomain" xlink:label="us-gaap_ClassOfWarrantOrRightDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfWarrantOrRightDomain" xlink:label="us-gaap_ClassOfWarrantOrRightDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfWarrantOrRightExercisePriceOfWarrantsOrRights1" xlink:label="us-gaap_ClassOfWarrantOrRightExercisePriceOfWarrantsOrRights1" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfWarrantOrRightNumberOfSecuritiesCalledByWarrantsOrRights" xlink:label="us-gaap_ClassOfWarrantOrRightNumberOfSecuritiesCalledByWarrantsOrRights" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DeferredOfferingCosts" xlink:label="us-gaap_DeferredOfferingCosts" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ProceedsFromIssuanceOrSaleOfEquity" xlink:label="us-gaap_ProceedsFromIssuanceOrSaleOfEquity" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SaleOfStockNameOfTransactionDomain" xlink:label="us-gaap_SaleOfStockNameOfTransactionDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SaleOfStockNameOfTransactionDomain" xlink:label="us-gaap_SaleOfStockNameOfTransactionDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ScheduleOfStockByClassTable" xlink:label="us-gaap_ScheduleOfStockByClassTable" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SharesIssuedPricePerShare" xlink:label="us-gaap_SharesIssuedPricePerShare" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StockIssuedDuringPeriodSharesNewIssues" xlink:label="us-gaap_StockIssuedDuringPeriodSharesNewIssues" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SubsidiarySaleOfStockAxis" xlink:label="us-gaap_SubsidiarySaleOfStockAxis" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_SubsidiarySaleOfStockAxis" xlink:to="us-gaap_SaleOfStockNameOfTransactionDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_ClassOfWarrantOrRightAxis" xlink:to="us-gaap_ClassOfWarrantOrRightDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_ScheduleOfStockByClassTable" xlink:to="us-gaap_SubsidiarySaleOfStockAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_SaleOfStockNameOfTransactionDomain" xlink:to="tlph_April2025PrivatePlacementThirdClosingMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_SubsidiarySaleOfStockAxis" xlink:to="us-gaap_SaleOfStockNameOfTransactionDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_ScheduleOfStockByClassTable" xlink:to="us-gaap_ClassOfWarrantOrRightAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfWarrantOrRightDomain" xlink:to="tlph_PrefundedWarrantsMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_ClassOfWarrantOrRightAxis" xlink:to="us-gaap_ClassOfWarrantOrRightDomain-default" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_ScheduleOfStockByClassTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ScheduleOfStockByClassTable" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="tlph_PrivatePlacementCovenantToTriggerSalePatientsEnrolled" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StockIssuedDuringPeriodSharesNewIssues" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_SharesIssuedPricePerShare" xlink:type="arc"/>
    <link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="tlph_ClassOfWarrantOrRightIssuedDuringPeriodPricePerWarrant" xlink:type="arc"/>
    <link:definitionArc order="5" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ClassOfWarrantOrRightNumberOfSecuritiesCalledByWarrantsOrRights" xlink:type="arc"/>
    <link:definitionArc order="6" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ClassOfWarrantOrRightExercisePriceOfWarrantsOrRights1" xlink:type="arc"/>
    <link:definitionArc order="7" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ProceedsFromIssuanceOrSaleOfEquity" xlink:type="arc"/>
    <link:definitionArc order="8" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DeferredOfferingCosts" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.talphera.com/20260331/role/statement-note-7-warrants-details-textual" xlink:href="tlph-20260331.xsd#statement-note-7-warrants-details-textual" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://www.talphera.com/20260331/role/statement-note-7-warrants-details-textual" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="tlph-20260331.xsd#tlph_April2025PrefundedWarrantsMember" xlink:label="tlph_April2025PrefundedWarrantsMember" xlink:type="locator"/>
    <link:loc xlink:href="tlph-20260331.xsd#tlph_ClassOfWarrantOrRightIssuedDuringPeriodPricePerWarrant" xlink:label="tlph_ClassOfWarrantOrRightIssuedDuringPeriodPricePerWarrant" xlink:type="locator"/>
    <link:loc xlink:href="tlph-20260331.xsd#tlph_ClassOfWarrantsOrRightExercisable" xlink:label="tlph_ClassOfWarrantsOrRightExercisable" xlink:type="locator"/>
    <link:loc xlink:href="tlph-20260331.xsd#tlph_PrefundWarrantsMember" xlink:label="tlph_PrefundWarrantsMember" xlink:type="locator"/>
    <link:loc xlink:href="tlph-20260331.xsd#tlph_PrefundedWarrantsMember" xlink:label="tlph_PrefundedWarrantsMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfWarrantOrRightAxis" xlink:label="us-gaap_ClassOfWarrantOrRightAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfWarrantOrRightDomain" xlink:label="us-gaap_ClassOfWarrantOrRightDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfWarrantOrRightDomain" xlink:label="us-gaap_ClassOfWarrantOrRightDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfWarrantOrRightExercisePriceOfWarrantsOrRights1" xlink:label="us-gaap_ClassOfWarrantOrRightExercisePriceOfWarrantsOrRights1" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfWarrantOrRightNumberOfSecuritiesCalledByWarrantsOrRights" xlink:label="us-gaap_ClassOfWarrantOrRightNumberOfSecuritiesCalledByWarrantsOrRights" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfWarrantOrRightOutstanding" xlink:label="us-gaap_ClassOfWarrantOrRightOutstanding" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfWarrantOrRightTable" xlink:label="us-gaap_ClassOfWarrantOrRightTable" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_ClassOfWarrantOrRightAxis" xlink:to="us-gaap_ClassOfWarrantOrRightDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_ClassOfWarrantOrRightTable" xlink:to="us-gaap_ClassOfWarrantOrRightAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfWarrantOrRightDomain" xlink:to="tlph_April2025PrefundedWarrantsMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_ClassOfWarrantOrRightAxis" xlink:to="us-gaap_ClassOfWarrantOrRightDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfWarrantOrRightDomain" xlink:to="tlph_PrefundedWarrantsMember" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfWarrantOrRightDomain" xlink:to="tlph_PrefundWarrantsMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_ClassOfWarrantOrRightTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ClassOfWarrantOrRightTable" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="tlph_ClassOfWarrantOrRightIssuedDuringPeriodPricePerWarrant" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ClassOfWarrantOrRightNumberOfSecuritiesCalledByWarrantsOrRights" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ClassOfWarrantOrRightExercisePriceOfWarrantsOrRights1" xlink:type="arc"/>
    <link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ClassOfWarrantOrRightOutstanding" xlink:type="arc"/>
    <link:definitionArc order="5" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="tlph_ClassOfWarrantsOrRightExercisable" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.talphera.com/20260331/role/statement-note-7-warrants-schedule-of-warrant-activity-details" xlink:href="tlph-20260331.xsd#statement-note-7-warrants-schedule-of-warrant-activity-details" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://www.talphera.com/20260331/role/statement-note-7-warrants-schedule-of-warrant-activity-details" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_RangeAxis" xlink:label="srt_RangeAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_RangeMember" xlink:label="srt_RangeMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_RangeMember" xlink:label="srt_RangeMember-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_WeightedAverageMember" xlink:label="srt_WeightedAverageMember" xlink:type="locator"/>
    <link:loc xlink:href="tlph-20260331.xsd#tlph_ClassOfWarrantOrRightExercisableDuringPeriodExercisePrice" xlink:label="tlph_ClassOfWarrantOrRightExercisableDuringPeriodExercisePrice" xlink:type="locator"/>
    <link:loc xlink:href="tlph-20260331.xsd#tlph_ClassOfWarrantOrRightIssued" xlink:label="tlph_ClassOfWarrantOrRightIssued" xlink:type="locator"/>
    <link:loc xlink:href="tlph-20260331.xsd#tlph_ClassOfWarrantOrRightIssuedExercisePrice" xlink:label="tlph_ClassOfWarrantOrRightIssuedExercisePrice" xlink:type="locator"/>
    <link:loc xlink:href="tlph-20260331.xsd#tlph_ClassOfWarrantsOrRightExercisable" xlink:label="tlph_ClassOfWarrantsOrRightExercisable" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfWarrantOrRightExercisePriceOfWarrantsOrRights1" xlink:label="us-gaap_ClassOfWarrantOrRightExercisePriceOfWarrantsOrRights1" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfWarrantOrRightOutstanding" xlink:label="us-gaap_ClassOfWarrantOrRightOutstanding" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfWarrantOrRightTable" xlink:label="us-gaap_ClassOfWarrantOrRightTable" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="srt_RangeAxis" xlink:to="srt_RangeMember" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_ClassOfWarrantOrRightTable" xlink:to="srt_RangeAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="srt_RangeMember" xlink:to="srt_WeightedAverageMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="srt_RangeAxis" xlink:to="srt_RangeMember-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_ClassOfWarrantOrRightTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ClassOfWarrantOrRightTable" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ClassOfWarrantOrRightOutstanding" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ClassOfWarrantOrRightExercisePriceOfWarrantsOrRights1" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="tlph_ClassOfWarrantOrRightIssued" xlink:type="arc"/>
    <link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="tlph_ClassOfWarrantOrRightIssuedExercisePrice" xlink:type="arc"/>
    <link:definitionArc order="5" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="tlph_ClassOfWarrantsOrRightExercisable" xlink:type="arc"/>
    <link:definitionArc order="6" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="tlph_ClassOfWarrantOrRightExercisableDuringPeriodExercisePrice" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.talphera.com/20260331/role/statement-note-8-stockbased-compensation-details-textual" xlink:href="tlph-20260331.xsd#statement-note-8-stockbased-compensation-details-textual" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://www.talphera.com/20260331/role/statement-note-8-stockbased-compensation-details-textual" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="tlph-20260331.xsd#tlph_AmendedEsppMember" xlink:label="tlph_AmendedEsppMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AwardTypeAxis" xlink:label="us-gaap_AwardTypeAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_EmployeeServiceShareBasedCompensationNonvestedAwardsTotalCompensationCostNotYetRecognized" xlink:label="us-gaap_EmployeeServiceShareBasedCompensationNonvestedAwardsTotalCompensationCostNotYetRecognized" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_EmployeeServiceShareBasedCompensationNonvestedAwardsTotalCompensationCostNotYetRecognizedPeriodForRecognition1" xlink:label="us-gaap_EmployeeServiceShareBasedCompensationNonvestedAwardsTotalCompensationCostNotYetRecognizedPeriodForRecognition1" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_PlanNameAxis" xlink:label="us-gaap_PlanNameAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_PlanNameDomain" xlink:label="us-gaap_PlanNameDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_PlanNameDomain" xlink:label="us-gaap_PlanNameDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_RestrictedStockUnitsRSUMember" xlink:label="us-gaap_RestrictedStockUnitsRSUMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ScheduleOfEmployeeServiceShareBasedCompensationAllocationOfRecognizedPeriodCostsTable" xlink:label="us-gaap_ScheduleOfEmployeeServiceShareBasedCompensationAllocationOfRecognizedPeriodCostsTable" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardNumberOfSharesAvailableForGrant" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardNumberOfSharesAvailableForGrant" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:label="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:label="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SharesPaidForTaxWithholdingForShareBasedCompensation" xlink:label="us-gaap_SharesPaidForTaxWithholdingForShareBasedCompensation" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_PlanNameAxis" xlink:to="us-gaap_PlanNameDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_AwardTypeAxis" xlink:to="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_ScheduleOfEmployeeServiceShareBasedCompensationAllocationOfRecognizedPeriodCostsTable" xlink:to="us-gaap_AwardTypeAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:to="us-gaap_RestrictedStockUnitsRSUMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_AwardTypeAxis" xlink:to="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_ScheduleOfEmployeeServiceShareBasedCompensationAllocationOfRecognizedPeriodCostsTable" xlink:to="us-gaap_PlanNameAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_PlanNameDomain" xlink:to="tlph_AmendedEsppMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_PlanNameAxis" xlink:to="us-gaap_PlanNameDomain-default" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_ScheduleOfEmployeeServiceShareBasedCompensationAllocationOfRecognizedPeriodCostsTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ScheduleOfEmployeeServiceShareBasedCompensationAllocationOfRecognizedPeriodCostsTable" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_SharesPaidForTaxWithholdingForShareBasedCompensation" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_EmployeeServiceShareBasedCompensationNonvestedAwardsTotalCompensationCostNotYetRecognized" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_EmployeeServiceShareBasedCompensationNonvestedAwardsTotalCompensationCostNotYetRecognizedPeriodForRecognition1" xlink:type="arc"/>
    <link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardNumberOfSharesAvailableForGrant" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.talphera.com/20260331/role/statement-note-8-stockbased-compensation-stockbased-compensation-expense-details" xlink:href="tlph-20260331.xsd#statement-note-8-stockbased-compensation-stockbased-compensation-expense-details" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://www.talphera.com/20260331/role/statement-note-8-stockbased-compensation-stockbased-compensation-expense-details" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AllocatedShareBasedCompensationExpense" xlink:label="us-gaap_AllocatedShareBasedCompensationExpense" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ScheduleOfEmployeeServiceShareBasedCompensationAllocationOfRecognizedPeriodCostsTable" xlink:label="us-gaap_ScheduleOfEmployeeServiceShareBasedCompensationAllocationOfRecognizedPeriodCostsTable" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_ScheduleOfEmployeeServiceShareBasedCompensationAllocationOfRecognizedPeriodCostsTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ScheduleOfEmployeeServiceShareBasedCompensationAllocationOfRecognizedPeriodCostsTable" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_AllocatedShareBasedCompensationExpense" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.talphera.com/20260331/role/statement-note-8-stockbased-compensation-stockbased-compensation-expense-2-details" xlink:href="tlph-20260331.xsd#statement-note-8-stockbased-compensation-stockbased-compensation-expense-2-details" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://www.talphera.com/20260331/role/statement-note-8-stockbased-compensation-stockbased-compensation-expense-2-details" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AllocatedShareBasedCompensationExpense" xlink:label="us-gaap_AllocatedShareBasedCompensationExpense" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ScheduleOfEmployeeServiceShareBasedCompensationAllocationOfRecognizedPeriodCostsTable" xlink:label="us-gaap_ScheduleOfEmployeeServiceShareBasedCompensationAllocationOfRecognizedPeriodCostsTable" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementOfIncomeLocationBalanceAxis" xlink:label="us-gaap_StatementOfIncomeLocationBalanceAxis" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_ScheduleOfEmployeeServiceShareBasedCompensationAllocationOfRecognizedPeriodCostsTable" xlink:to="us-gaap_StatementOfIncomeLocationBalanceAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_ScheduleOfEmployeeServiceShareBasedCompensationAllocationOfRecognizedPeriodCostsTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ScheduleOfEmployeeServiceShareBasedCompensationAllocationOfRecognizedPeriodCostsTable" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_AllocatedShareBasedCompensationExpense" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.talphera.com/20260331/role/statement-note-8-stockbased-compensation-restricted-stock-activity-details" xlink:href="tlph-20260331.xsd#statement-note-8-stockbased-compensation-restricted-stock-activity-details" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://www.talphera.com/20260331/role/statement-note-8-stockbased-compensation-restricted-stock-activity-details" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AwardTypeAxis" xlink:label="us-gaap_AwardTypeAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_RestrictedStockUnitsRSUMember" xlink:label="us-gaap_RestrictedStockUnitsRSUMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ScheduleOfEmployeeServiceShareBasedCompensationAllocationOfRecognizedPeriodCostsTable" xlink:label="us-gaap_ScheduleOfEmployeeServiceShareBasedCompensationAllocationOfRecognizedPeriodCostsTable" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsForfeitedInPeriod" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsForfeitedInPeriod" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsForfeituresWeightedAverageGrantDateFairValue" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsForfeituresWeightedAverageGrantDateFairValue" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsGrantsInPeriod" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsGrantsInPeriod" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsGrantsInPeriodWeightedAverageGrantDateFairValue" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsGrantsInPeriodWeightedAverageGrantDateFairValue" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedNumber" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedNumber" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedWeightedAverageGrantDateFairValue" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedWeightedAverageGrantDateFairValue" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsVestedInPeriod" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsVestedInPeriod" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsVestedInPeriodWeightedAverageGrantDateFairValue" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsVestedInPeriodWeightedAverageGrantDateFairValue" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:label="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:label="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_AwardTypeAxis" xlink:to="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_ScheduleOfEmployeeServiceShareBasedCompensationAllocationOfRecognizedPeriodCostsTable" xlink:to="us-gaap_AwardTypeAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:to="us-gaap_RestrictedStockUnitsRSUMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_AwardTypeAxis" xlink:to="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_ScheduleOfEmployeeServiceShareBasedCompensationAllocationOfRecognizedPeriodCostsTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ScheduleOfEmployeeServiceShareBasedCompensationAllocationOfRecognizedPeriodCostsTable" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedNumber" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedWeightedAverageGrantDateFairValue" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsGrantsInPeriod" xlink:type="arc"/>
    <link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsGrantsInPeriodWeightedAverageGrantDateFairValue" xlink:type="arc"/>
    <link:definitionArc order="5" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsVestedInPeriod" xlink:type="arc"/>
    <link:definitionArc order="6" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsVestedInPeriodWeightedAverageGrantDateFairValue" xlink:type="arc"/>
    <link:definitionArc order="7" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsForfeitedInPeriod" xlink:type="arc"/>
    <link:definitionArc order="8" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsForfeituresWeightedAverageGrantDateFairValue" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.talphera.com/20260331/role/statement-note-8-stockbased-compensation-option-activity-details" xlink:href="tlph-20260331.xsd#statement-note-8-stockbased-compensation-option-activity-details" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://www.talphera.com/20260331/role/statement-note-8-stockbased-compensation-option-activity-details" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="tlph-20260331.xsd#tlph_SharebasedCompensationArrangementBySharebasedPaymentAwardOptionsExercisableAndVestedNumber" xlink:label="tlph_SharebasedCompensationArrangementBySharebasedPaymentAwardOptionsExercisableAndVestedNumber" xlink:type="locator"/>
    <link:loc xlink:href="tlph-20260331.xsd#tlph_SharebasedCompensationArrangementBySharebasedPaymentAwardOptionsVestedAndExercisableWeightedAverageRemainingContractualTerm" xlink:label="tlph_SharebasedCompensationArrangementBySharebasedPaymentAwardOptionsVestedAndExercisableWeightedAverageRemainingContractualTerm" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ScheduleOfEmployeeServiceShareBasedCompensationAllocationOfRecognizedPeriodCostsTable" xlink:label="us-gaap_ScheduleOfEmployeeServiceShareBasedCompensationAllocationOfRecognizedPeriodCostsTable" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsExpirationsInPeriod" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsExpirationsInPeriod" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsForfeituresInPeriod" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsForfeituresInPeriod" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsGrantsInPeriodGross" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsGrantsInPeriodGross" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingIntrinsicValue" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingIntrinsicValue" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingNumber" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingNumber" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingWeightedAverageExercisePrice" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingWeightedAverageExercisePrice" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsVestedAndExpectedToVestExercisableAggregateIntrinsicValue" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsVestedAndExpectedToVestExercisableAggregateIntrinsicValue" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsVestedAndExpectedToVestExercisableWeightedAverageExercisePrice" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsVestedAndExpectedToVestExercisableWeightedAverageExercisePrice" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsVestedAndExpectedToVestOutstandingAggregateIntrinsicValue" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsVestedAndExpectedToVestOutstandingAggregateIntrinsicValue" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsVestedAndExpectedToVestOutstandingNumber" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsVestedAndExpectedToVestOutstandingNumber" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsVestedAndExpectedToVestOutstandingWeightedAverageExercisePrice" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsVestedAndExpectedToVestOutstandingWeightedAverageExercisePrice" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardOptionsExercisesInPeriodWeightedAverageExercisePrice" xlink:label="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardOptionsExercisesInPeriodWeightedAverageExercisePrice" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardOptionsExpirationsInPeriodWeightedAverageExercisePrice" xlink:label="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardOptionsExpirationsInPeriodWeightedAverageExercisePrice" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardOptionsForfeituresInPeriodWeightedAverageExercisePrice" xlink:label="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardOptionsForfeituresInPeriodWeightedAverageExercisePrice" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardOptionsGrantsInPeriodWeightedAverageExercisePrice" xlink:label="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardOptionsGrantsInPeriodWeightedAverageExercisePrice" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardOptionsOutstandingWeightedAverageRemainingContractualTerm2" xlink:label="us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardOptionsOutstandingWeightedAverageRemainingContractualTerm2" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardOptionsVestedAndExpectedToVestOutstandingWeightedAverageRemainingContractualTerm1" xlink:label="us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardOptionsVestedAndExpectedToVestOutstandingWeightedAverageRemainingContractualTerm1" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StockIssuedDuringPeriodSharesStockOptionsExercised" xlink:label="us-gaap_StockIssuedDuringPeriodSharesStockOptionsExercised" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_ScheduleOfEmployeeServiceShareBasedCompensationAllocationOfRecognizedPeriodCostsTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ScheduleOfEmployeeServiceShareBasedCompensationAllocationOfRecognizedPeriodCostsTable" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingNumber" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingWeightedAverageExercisePrice" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsGrantsInPeriodGross" xlink:type="arc"/>
    <link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardOptionsGrantsInPeriodWeightedAverageExercisePrice" xlink:type="arc"/>
    <link:definitionArc order="5" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsForfeituresInPeriod" xlink:type="arc"/>
    <link:definitionArc order="6" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardOptionsForfeituresInPeriodWeightedAverageExercisePrice" xlink:type="arc"/>
    <link:definitionArc order="7" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsExpirationsInPeriod" xlink:type="arc"/>
    <link:definitionArc order="8" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardOptionsExpirationsInPeriodWeightedAverageExercisePrice" xlink:type="arc"/>
    <link:definitionArc order="9" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StockIssuedDuringPeriodSharesStockOptionsExercised" xlink:type="arc"/>
    <link:definitionArc order="10" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardOptionsExercisesInPeriodWeightedAverageExercisePrice" xlink:type="arc"/>
    <link:definitionArc order="11" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardOptionsOutstandingWeightedAverageRemainingContractualTerm2" xlink:type="arc"/>
    <link:definitionArc order="12" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingIntrinsicValue" xlink:type="arc"/>
    <link:definitionArc order="13" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="tlph_SharebasedCompensationArrangementBySharebasedPaymentAwardOptionsExercisableAndVestedNumber" xlink:type="arc"/>
    <link:definitionArc order="14" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsVestedAndExpectedToVestExercisableWeightedAverageExercisePrice" xlink:type="arc"/>
    <link:definitionArc order="15" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="tlph_SharebasedCompensationArrangementBySharebasedPaymentAwardOptionsVestedAndExercisableWeightedAverageRemainingContractualTerm" xlink:type="arc"/>
    <link:definitionArc order="16" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsVestedAndExpectedToVestExercisableAggregateIntrinsicValue" xlink:type="arc"/>
    <link:definitionArc order="17" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsVestedAndExpectedToVestOutstandingNumber" xlink:type="arc"/>
    <link:definitionArc order="18" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsVestedAndExpectedToVestOutstandingWeightedAverageExercisePrice" xlink:type="arc"/>
    <link:definitionArc order="19" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardOptionsVestedAndExpectedToVestOutstandingWeightedAverageRemainingContractualTerm1" xlink:type="arc"/>
    <link:definitionArc order="20" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsVestedAndExpectedToVestOutstandingAggregateIntrinsicValue" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.talphera.com/20260331/role/statement-note-9-net-loss-per-share-of-common-stock-details-textual" xlink:href="tlph-20260331.xsd#statement-note-9-net-loss-per-share-of-common-stock-details-textual" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://www.talphera.com/20260331/role/statement-note-9-net-loss-per-share-of-common-stock-details-textual" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="tlph-20260331.xsd#tlph_April2025PrefundedWarrantsMember" xlink:label="tlph_April2025PrefundedWarrantsMember" xlink:type="locator"/>
    <link:loc xlink:href="tlph-20260331.xsd#tlph_January2024PrefundedWarrantsMember" xlink:label="tlph_January2024PrefundedWarrantsMember" xlink:type="locator"/>
    <link:loc xlink:href="tlph-20260331.xsd#tlph_July2023PrefundedWarrantsMember" xlink:label="tlph_July2023PrefundedWarrantsMember" xlink:type="locator"/>
    <link:loc xlink:href="tlph-20260331.xsd#tlph_September2025PrefundedWarrantsMember" xlink:label="tlph_September2025PrefundedWarrantsMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfWarrantOrRightAxis" xlink:label="us-gaap_ClassOfWarrantOrRightAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfWarrantOrRightDomain" xlink:label="us-gaap_ClassOfWarrantOrRightDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfWarrantOrRightDomain" xlink:label="us-gaap_ClassOfWarrantOrRightDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfWarrantOrRightExercisePriceOfWarrantsOrRights1" xlink:label="us-gaap_ClassOfWarrantOrRightExercisePriceOfWarrantsOrRights1" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ScheduleOfEarningsPerShareBasicByCommonClassTable" xlink:label="us-gaap_ScheduleOfEarningsPerShareBasicByCommonClassTable" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_ClassOfWarrantOrRightAxis" xlink:to="us-gaap_ClassOfWarrantOrRightDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_ScheduleOfEarningsPerShareBasicByCommonClassTable" xlink:to="us-gaap_ClassOfWarrantOrRightAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfWarrantOrRightDomain" xlink:to="tlph_September2025PrefundedWarrantsMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_ClassOfWarrantOrRightAxis" xlink:to="us-gaap_ClassOfWarrantOrRightDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfWarrantOrRightDomain" xlink:to="tlph_April2025PrefundedWarrantsMember" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfWarrantOrRightDomain" xlink:to="tlph_January2024PrefundedWarrantsMember" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfWarrantOrRightDomain" xlink:to="tlph_July2023PrefundedWarrantsMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_ScheduleOfEarningsPerShareBasicByCommonClassTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ScheduleOfEarningsPerShareBasicByCommonClassTable" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ClassOfWarrantOrRightExercisePriceOfWarrantsOrRights1" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.talphera.com/20260331/role/statement-note-9-net-loss-per-share-of-common-stock-common-stock-excluded-from-computation-of-diluted-net-loss-per-share-details" xlink:href="tlph-20260331.xsd#statement-note-9-net-loss-per-share-of-common-stock-common-stock-excluded-from-computation-of-diluted-net-loss-per-share-details" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://www.talphera.com/20260331/role/statement-note-9-net-loss-per-share-of-common-stock-common-stock-excluded-from-computation-of-diluted-net-loss-per-share-details" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="tlph-20260331.xsd#tlph_RSUsESPPAndEmployeeStockOptionsMember" xlink:label="tlph_RSUsESPPAndEmployeeStockOptionsMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareAmount" xlink:label="us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareAmount" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareByAntidilutiveSecuritiesAxis" xlink:label="us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareByAntidilutiveSecuritiesAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AntidilutiveSecuritiesNameDomain" xlink:label="us-gaap_AntidilutiveSecuritiesNameDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AntidilutiveSecuritiesNameDomain" xlink:label="us-gaap_AntidilutiveSecuritiesNameDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ScheduleOfEarningsPerShareBasicByCommonClassTable" xlink:label="us-gaap_ScheduleOfEarningsPerShareBasicByCommonClassTable" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_WarrantMember" xlink:label="us-gaap_WarrantMember" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareByAntidilutiveSecuritiesAxis" xlink:to="us-gaap_AntidilutiveSecuritiesNameDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_ScheduleOfEarningsPerShareBasicByCommonClassTable" xlink:to="us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareByAntidilutiveSecuritiesAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AntidilutiveSecuritiesNameDomain" xlink:to="tlph_RSUsESPPAndEmployeeStockOptionsMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareByAntidilutiveSecuritiesAxis" xlink:to="us-gaap_AntidilutiveSecuritiesNameDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AntidilutiveSecuritiesNameDomain" xlink:to="us-gaap_WarrantMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_ScheduleOfEarningsPerShareBasicByCommonClassTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ScheduleOfEarningsPerShareBasicByCommonClassTable" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareAmount" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.talphera.com/20260331/role/statement-note-10-segment-information-details-textual" xlink:href="tlph-20260331.xsd#statement-note-10-segment-information-details-textual" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://www.talphera.com/20260331/role/statement-note-10-segment-information-details-textual" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_NumberOfReportableSegments" xlink:label="us-gaap_NumberOfReportableSegments" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ScheduleOfSegmentReportingInformationBySegmentTable" xlink:label="us-gaap_ScheduleOfSegmentReportingInformationBySegmentTable" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_ScheduleOfSegmentReportingInformationBySegmentTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ScheduleOfSegmentReportingInformationBySegmentTable" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_NumberOfReportableSegments" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.talphera.com/20260331/role/statement-note-10-segment-information-summary-of-segment-loss-details" xlink:href="tlph-20260331.xsd#statement-note-10-segment-information-summary-of-segment-loss-details" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://www.talphera.com/20260331/role/statement-note-10-segment-information-summary-of-segment-loss-details" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="tlph-20260331.xsd#tlph_DevelopmentAndClinicalTrialExpense" xlink:label="tlph_DevelopmentAndClinicalTrialExpense" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_IncomeLossFromDiscontinuedOperationsNetOfTaxAttributableToReportingEntity" xlink:label="us-gaap_IncomeLossFromDiscontinuedOperationsNetOfTaxAttributableToReportingEntity" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_LaborAndRelatedExpense" xlink:label="us-gaap_LaborAndRelatedExpense" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_NetIncomeLoss" xlink:label="us-gaap_NetIncomeLoss" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_OtherSellingGeneralAndAdministrativeExpense" xlink:label="us-gaap_OtherSellingGeneralAndAdministrativeExpense" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_RevenueFromContractWithCustomerIncludingAssessedTax" xlink:label="us-gaap_RevenueFromContractWithCustomerIncludingAssessedTax" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ScheduleOfSegmentReportingInformationBySegmentTable" xlink:label="us-gaap_ScheduleOfSegmentReportingInformationBySegmentTable" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SegmentReportingOtherItemAmount" xlink:label="us-gaap_SegmentReportingOtherItemAmount" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_ScheduleOfSegmentReportingInformationBySegmentTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ScheduleOfSegmentReportingInformationBySegmentTable" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_RevenueFromContractWithCustomerIncludingAssessedTax" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_LaborAndRelatedExpense" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="tlph_DevelopmentAndClinicalTrialExpense" xlink:type="arc"/>
    <link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_OtherSellingGeneralAndAdministrativeExpense" xlink:type="arc"/>
    <link:definitionArc order="5" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_SegmentReportingOtherItemAmount" xlink:type="arc"/>
    <link:definitionArc order="6" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_IncomeLossFromDiscontinuedOperationsNetOfTaxAttributableToReportingEntity" xlink:type="arc"/>
    <link:definitionArc order="7" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_NetIncomeLoss" xlink:type="arc"/>
  </link:definitionLink>
</link:linkbase>
