<?xml version="1.0" encoding="US-ASCII" standalone="no"?>
    <!-- Field: Doc-Info; Name: Generator; Value: GoFiler Complete; Version: 6.5a -->
    <!-- Field: Doc-Info; Name: VendorURI; Value: https://www.novaworks.com -->
    <!-- Field: Doc-Info; Name: Status; Value: 0x00000000 -->
<link:linkbase xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" xmlns:xlink="http://www.w3.org/1999/xlink" xmlns:link="http://www.xbrl.org/2003/linkbase" xmlns:xbrldt="http://xbrl.org/2005/xbrldt" xmlns:xbrli="http://www.xbrl.org/2003/instance" xsi:schemaLocation="http://www.xbrl.org/2003/linkbase http://www.xbrl.org/2003/xbrl-linkbase-2003-12-31.xsd">
    <link:roleRef xlink:type="simple" xlink:href="ptix-20260630.xsd#Cover" roleURI="http://protagenic.com/role/Cover" />
    <link:roleRef xlink:type="simple" xlink:href="ptix-20260630.xsd#BalanceSheets" roleURI="http://protagenic.com/role/BalanceSheets" />
    <link:roleRef xlink:type="simple" xlink:href="ptix-20260630.xsd#BalanceSheetsParenthetical" roleURI="http://protagenic.com/role/BalanceSheetsParenthetical" />
    <link:roleRef xlink:type="simple" xlink:href="ptix-20260630.xsd#StatementsOfOperationsAndComprehensiveIncomeLoss" roleURI="http://protagenic.com/role/StatementsOfOperationsAndComprehensiveIncomeLoss" />
    <link:roleRef xlink:type="simple" xlink:href="ptix-20260630.xsd#StatementsOfChangeInStockholdersEquity" roleURI="http://protagenic.com/role/StatementsOfChangeInStockholdersEquity" />
    <link:roleRef xlink:type="simple" xlink:href="ptix-20260630.xsd#StatementsOfCashFlows" roleURI="http://protagenic.com/role/StatementsOfCashFlows" />
    <link:roleRef xlink:type="simple" xlink:href="ptix-20260630.xsd#OrganizationAndNatureOfBusiness" roleURI="http://protagenic.com/role/OrganizationAndNatureOfBusiness" />
    <link:roleRef xlink:type="simple" xlink:href="ptix-20260630.xsd#LiquidityAndGoingConcern" roleURI="http://protagenic.com/role/LiquidityAndGoingConcern" />
    <link:roleRef xlink:type="simple" xlink:href="ptix-20260630.xsd#SummaryOfSignificantAccountingPolicies" roleURI="http://protagenic.com/role/SummaryOfSignificantAccountingPolicies" />
    <link:roleRef xlink:type="simple" xlink:href="ptix-20260630.xsd#ReverseMerger" roleURI="http://protagenic.com/role/ReverseMerger" />
    <link:roleRef xlink:type="simple" xlink:href="ptix-20260630.xsd#FixedAssets" roleURI="http://protagenic.com/role/FixedAssets" />
    <link:roleRef xlink:type="simple" xlink:href="ptix-20260630.xsd#IntangibleAssets" roleURI="http://protagenic.com/role/IntangibleAssets" />
    <link:roleRef xlink:type="simple" xlink:href="ptix-20260630.xsd#AccountsPayable" roleURI="http://protagenic.com/role/AccountsPayable" />
    <link:roleRef xlink:type="simple" xlink:href="ptix-20260630.xsd#AccruedLiabilities" roleURI="http://protagenic.com/role/AccruedLiabilities" />
    <link:roleRef xlink:type="simple" xlink:href="ptix-20260630.xsd#RelatedPartyTransactions" roleURI="http://protagenic.com/role/RelatedPartyTransactions" />
    <link:roleRef xlink:type="simple" xlink:href="ptix-20260630.xsd#StockholdersEquity" roleURI="http://protagenic.com/role/StockholdersEquity" />
    <link:roleRef xlink:type="simple" xlink:href="ptix-20260630.xsd#CollaborativeAgreements" roleURI="http://protagenic.com/role/CollaborativeAgreements" />
    <link:roleRef xlink:type="simple" xlink:href="ptix-20260630.xsd#CommitmentsAndContingencies" roleURI="http://protagenic.com/role/CommitmentsAndContingencies" />
    <link:roleRef xlink:type="simple" xlink:href="ptix-20260630.xsd#SegmentReporting" roleURI="http://protagenic.com/role/SegmentReporting" />
    <link:roleRef xlink:type="simple" xlink:href="ptix-20260630.xsd#SubsequentEvents" roleURI="http://protagenic.com/role/SubsequentEvents" />
    <link:roleRef xlink:type="simple" xlink:href="ptix-20260630.xsd#SummaryOfSignificantAccountingPoliciesPolicies" roleURI="http://protagenic.com/role/SummaryOfSignificantAccountingPoliciesPolicies" />
    <link:roleRef xlink:type="simple" xlink:href="ptix-20260630.xsd#SummaryOfSignificantAccountingPoliciesTables" roleURI="http://protagenic.com/role/SummaryOfSignificantAccountingPoliciesTables" />
    <link:roleRef xlink:type="simple" xlink:href="ptix-20260630.xsd#ReverseMergerTables" roleURI="http://protagenic.com/role/ReverseMergerTables" />
    <link:roleRef xlink:type="simple" xlink:href="ptix-20260630.xsd#FixedAssetsTables" roleURI="http://protagenic.com/role/FixedAssetsTables" />
    <link:roleRef xlink:type="simple" xlink:href="ptix-20260630.xsd#IntangibleAssetsTables" roleURI="http://protagenic.com/role/IntangibleAssetsTables" />
    <link:roleRef xlink:type="simple" xlink:href="ptix-20260630.xsd#AccountsPayableTables" roleURI="http://protagenic.com/role/AccountsPayableTables" />
    <link:roleRef xlink:type="simple" xlink:href="ptix-20260630.xsd#AccruedLiabilitiesTables" roleURI="http://protagenic.com/role/AccruedLiabilitiesTables" />
    <link:roleRef xlink:type="simple" xlink:href="ptix-20260630.xsd#StockholdersEquityTables" roleURI="http://protagenic.com/role/StockholdersEquityTables" />
    <link:roleRef xlink:type="simple" xlink:href="ptix-20260630.xsd#OrganizationAndNatureOfBusinessDetailsNarrative" roleURI="http://protagenic.com/role/OrganizationAndNatureOfBusinessDetailsNarrative" />
    <link:roleRef xlink:type="simple" xlink:href="ptix-20260630.xsd#LiquidityAndGoingConcernDetailsNarrative" roleURI="http://protagenic.com/role/LiquidityAndGoingConcernDetailsNarrative" />
    <link:roleRef xlink:type="simple" xlink:href="ptix-20260630.xsd#ScheduleOfAnti-dilutiveSecuritiesExcludedFromComputationOfEarningsPerShareDetails" roleURI="http://protagenic.com/role/ScheduleOfAnti-dilutiveSecuritiesExcludedFromComputationOfEarningsPerShareDetails" />
    <link:roleRef xlink:type="simple" xlink:href="ptix-20260630.xsd#SummaryOfSignificantAccountingPoliciesDetailsNarrative" roleURI="http://protagenic.com/role/SummaryOfSignificantAccountingPoliciesDetailsNarrative" />
    <link:roleRef xlink:type="simple" xlink:href="ptix-20260630.xsd#ScheduleOfPurchasePriceAllocationDetails" roleURI="http://protagenic.com/role/ScheduleOfPurchasePriceAllocationDetails" />
    <link:roleRef xlink:type="simple" xlink:href="ptix-20260630.xsd#ScheduleOfBusinessCombinationContingentConsiderationDetails" roleURI="http://protagenic.com/role/ScheduleOfBusinessCombinationContingentConsiderationDetails" />
    <link:roleRef xlink:type="simple" xlink:href="ptix-20260630.xsd#ReverseMergerDetailsNarrative" roleURI="http://protagenic.com/role/ReverseMergerDetailsNarrative" />
    <link:roleRef xlink:type="simple" xlink:href="ptix-20260630.xsd#ScheduleOfFixedAssetsDetails" roleURI="http://protagenic.com/role/ScheduleOfFixedAssetsDetails" />
    <link:roleRef xlink:type="simple" xlink:href="ptix-20260630.xsd#FixedAssetsDetailsNarrative" roleURI="http://protagenic.com/role/FixedAssetsDetailsNarrative" />
    <link:roleRef xlink:type="simple" xlink:href="ptix-20260630.xsd#ScheduleOfIntangibleAssetsDetails" roleURI="http://protagenic.com/role/ScheduleOfIntangibleAssetsDetails" />
    <link:roleRef xlink:type="simple" xlink:href="ptix-20260630.xsd#IntangibleAssetsDetailsNarrative" roleURI="http://protagenic.com/role/IntangibleAssetsDetailsNarrative" />
    <link:roleRef xlink:type="simple" xlink:href="ptix-20260630.xsd#ScheduleOfAccountsPayableDetails" roleURI="http://protagenic.com/role/ScheduleOfAccountsPayableDetails" />
    <link:roleRef xlink:type="simple" xlink:href="ptix-20260630.xsd#ScheduleOfAccruedLiabilitiesDetails" roleURI="http://protagenic.com/role/ScheduleOfAccruedLiabilitiesDetails" />
    <link:roleRef xlink:type="simple" xlink:href="ptix-20260630.xsd#RelatedPartyTransactionsDetailsNarrative" roleURI="http://protagenic.com/role/RelatedPartyTransactionsDetailsNarrative" />
    <link:roleRef xlink:type="simple" xlink:href="ptix-20260630.xsd#ScheduleOfFairValueOfEachOptionGrantedDetails" roleURI="http://protagenic.com/role/ScheduleOfFairValueOfEachOptionGrantedDetails" />
    <link:roleRef xlink:type="simple" xlink:href="ptix-20260630.xsd#ScheduleOfShare-basedCompensationStockOptionsActivityDetails" roleURI="http://protagenic.com/role/ScheduleOfShare-basedCompensationStockOptionsActivityDetails" />
    <link:roleRef xlink:type="simple" xlink:href="ptix-20260630.xsd#ScheduleOfShare-basedCompensationNonvestedSharesDetails" roleURI="http://protagenic.com/role/ScheduleOfShare-basedCompensationNonvestedSharesDetails" />
    <link:roleRef xlink:type="simple" xlink:href="ptix-20260630.xsd#SummaryOfWarrantDetails" roleURI="http://protagenic.com/role/SummaryOfWarrantDetails" />
    <link:roleRef xlink:type="simple" xlink:href="ptix-20260630.xsd#StockholdersEquityDetailsNarrative" roleURI="http://protagenic.com/role/StockholdersEquityDetailsNarrative" />
    <link:roleRef xlink:type="simple" xlink:href="ptix-20260630.xsd#CollaborativeAgreementsDetailsNarrative" roleURI="http://protagenic.com/role/CollaborativeAgreementsDetailsNarrative" />
    <link:roleRef xlink:type="simple" xlink:href="ptix-20260630.xsd#CommitmentsAndContingenciesDetailsNarrative" roleURI="http://protagenic.com/role/CommitmentsAndContingenciesDetailsNarrative" />
    <link:roleRef xlink:type="simple" xlink:href="ptix-20260630.xsd#SegmentReportingDetailsNarrative" roleURI="http://protagenic.com/role/SegmentReportingDetailsNarrative" />
    <link:arcroleRef xlink:type="simple" xlink:href="http://www.xbrl.org/2005/xbrldt-2005.xsd#hypercube-dimension" arcroleURI="http://xbrl.org/int/dim/arcrole/hypercube-dimension" />
    <link:arcroleRef xlink:type="simple" xlink:href="http://www.xbrl.org/2005/xbrldt-2005.xsd#dimension-domain" arcroleURI="http://xbrl.org/int/dim/arcrole/dimension-domain" />
    <link:arcroleRef xlink:type="simple" xlink:href="http://www.xbrl.org/2005/xbrldt-2005.xsd#domain-member" arcroleURI="http://xbrl.org/int/dim/arcrole/domain-member" />
    <link:arcroleRef xlink:type="simple" xlink:href="http://www.xbrl.org/2005/xbrldt-2005.xsd#all" arcroleURI="http://xbrl.org/int/dim/arcrole/all" />
    <link:arcroleRef xlink:type="simple" xlink:href="http://www.xbrl.org/2005/xbrldt-2005.xsd#notAll" arcroleURI="http://xbrl.org/int/dim/arcrole/notAll" />
    <link:arcroleRef xlink:type="simple" xlink:href="http://www.xbrl.org/2005/xbrldt-2005.xsd#dimension-default" arcroleURI="http://xbrl.org/int/dim/arcrole/dimension-default" />
    <link:definitionLink xlink:type="extended" xlink:role="http://protagenic.com/role/Cover" xlink:title="00000001 - Document - Cover">
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementLineItems" xlink:label="loc_us-gaapStatementLineItems" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementTable" xlink:label="loc_us-gaapStatementTable" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xbrldt:contextElement="segment" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapStatementTable" xlink:type="arc" order="10" xbrldt:closed="true" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementEquityComponentsAxis" xlink:label="loc_us-gaapStatementEquityComponentsAxis" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaapStatementTable" xlink:to="loc_us-gaapStatementEquityComponentsAxis" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_EquityComponentDomain" xlink:label="loc_us-gaapEquityComponentDomain" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaapStatementEquityComponentsAxis" xlink:to="loc_us-gaapEquityComponentDomain" xlink:type="arc" order="20" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_EquityComponentDomain" xlink:label="loc_us-gaapEquityComponentDomain_10" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xbrldt:contextElement="segment" xlink:from="loc_us-gaapStatementEquityComponentsAxis" xlink:to="loc_us-gaapEquityComponentDomain_10" xlink:type="arc" order="30" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_CommonStockMember" xlink:label="loc_us-gaapCommonStockMember_10" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapEquityComponentDomain" xlink:to="loc_us-gaapCommonStockMember_10" xlink:type="arc" order="11" />
      <link:loc xlink:type="locator" xlink:href="ptix-20260630.xsd#PTIX_CommonStockPurchaseWarrantMember" xlink:label="loc_PTIXCommonStockPurchaseWarrantMember_20" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapEquityComponentDomain" xlink:to="loc_PTIXCommonStockPurchaseWarrantMember_20" xlink:type="arc" order="21" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_DocumentType" xlink:label="loc_deiDocumentType_50" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_deiDocumentType_50" xlink:type="arc" order="0" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_AmendmentFlag" xlink:label="loc_deiAmendmentFlag_50" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_deiAmendmentFlag_50" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_AmendmentDescription" xlink:label="loc_deiAmendmentDescription_50" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_deiAmendmentDescription_50" xlink:type="arc" order="2" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_DocumentRegistrationStatement" xlink:label="loc_deiDocumentRegistrationStatement_50" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_deiDocumentRegistrationStatement_50" xlink:type="arc" order="3" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_DocumentAnnualReport" xlink:label="loc_deiDocumentAnnualReport_50" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_deiDocumentAnnualReport_50" xlink:type="arc" order="4" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_DocumentQuarterlyReport" xlink:label="loc_deiDocumentQuarterlyReport_50" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_deiDocumentQuarterlyReport_50" xlink:type="arc" order="5" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_DocumentTransitionReport" xlink:label="loc_deiDocumentTransitionReport_50" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_deiDocumentTransitionReport_50" xlink:type="arc" order="6" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_DocumentShellCompanyReport" xlink:label="loc_deiDocumentShellCompanyReport_50" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_deiDocumentShellCompanyReport_50" xlink:type="arc" order="7" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_DocumentShellCompanyEventDate" xlink:label="loc_deiDocumentShellCompanyEventDate_50" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_deiDocumentShellCompanyEventDate_50" xlink:type="arc" order="8" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_DocumentPeriodStartDate" xlink:label="loc_deiDocumentPeriodStartDate_50" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_deiDocumentPeriodStartDate_50" xlink:type="arc" order="9" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_DocumentPeriodEndDate" xlink:label="loc_deiDocumentPeriodEndDate_50" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_deiDocumentPeriodEndDate_50" xlink:type="arc" order="10" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_DocumentFiscalPeriodFocus" xlink:label="loc_deiDocumentFiscalPeriodFocus_50" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_deiDocumentFiscalPeriodFocus_50" xlink:type="arc" order="11" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_DocumentFiscalYearFocus" xlink:label="loc_deiDocumentFiscalYearFocus_50" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_deiDocumentFiscalYearFocus_50" xlink:type="arc" order="12" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_CurrentFiscalYearEndDate" xlink:label="loc_deiCurrentFiscalYearEndDate_50" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_deiCurrentFiscalYearEndDate_50" xlink:type="arc" order="13" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityFileNumber" xlink:label="loc_deiEntityFileNumber_50" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_deiEntityFileNumber_50" xlink:type="arc" order="14" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityRegistrantName" xlink:label="loc_deiEntityRegistrantName_50" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_deiEntityRegistrantName_50" xlink:type="arc" order="15" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityCentralIndexKey" xlink:label="loc_deiEntityCentralIndexKey_50" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_deiEntityCentralIndexKey_50" xlink:type="arc" order="16" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityPrimarySicNumber" xlink:label="loc_deiEntityPrimarySicNumber_50" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_deiEntityPrimarySicNumber_50" xlink:type="arc" order="17" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityTaxIdentificationNumber" xlink:label="loc_deiEntityTaxIdentificationNumber_50" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_deiEntityTaxIdentificationNumber_50" xlink:type="arc" order="18" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityIncorporationStateCountryCode" xlink:label="loc_deiEntityIncorporationStateCountryCode_50" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_deiEntityIncorporationStateCountryCode_50" xlink:type="arc" order="19" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityAddressAddressLine1" xlink:label="loc_deiEntityAddressAddressLine1_50" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_deiEntityAddressAddressLine1_50" xlink:type="arc" order="20" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityAddressAddressLine2" xlink:label="loc_deiEntityAddressAddressLine2_50" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_deiEntityAddressAddressLine2_50" xlink:type="arc" order="21" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityAddressAddressLine3" xlink:label="loc_deiEntityAddressAddressLine3_50" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_deiEntityAddressAddressLine3_50" xlink:type="arc" order="22" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityAddressCityOrTown" xlink:label="loc_deiEntityAddressCityOrTown_50" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_deiEntityAddressCityOrTown_50" xlink:type="arc" order="23" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityAddressStateOrProvince" xlink:label="loc_deiEntityAddressStateOrProvince_50" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_deiEntityAddressStateOrProvince_50" xlink:type="arc" order="24" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityAddressCountry" xlink:label="loc_deiEntityAddressCountry_50" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_deiEntityAddressCountry_50" xlink:type="arc" order="25" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityAddressPostalZipCode" xlink:label="loc_deiEntityAddressPostalZipCode_50" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_deiEntityAddressPostalZipCode_50" xlink:type="arc" order="26" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_CountryRegion" xlink:label="loc_deiCountryRegion_50" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_deiCountryRegion_50" xlink:type="arc" order="27" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_CityAreaCode" xlink:label="loc_deiCityAreaCode_50" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_deiCityAreaCode_50" xlink:type="arc" order="28" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_LocalPhoneNumber" xlink:label="loc_deiLocalPhoneNumber_50" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_deiLocalPhoneNumber_50" xlink:type="arc" order="29" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_Extension" xlink:label="loc_deiExtension_50" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_deiExtension_50" xlink:type="arc" order="30" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_WrittenCommunications" xlink:label="loc_deiWrittenCommunications_50" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_deiWrittenCommunications_50" xlink:type="arc" order="31" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_SolicitingMaterial" xlink:label="loc_deiSolicitingMaterial_50" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_deiSolicitingMaterial_50" xlink:type="arc" order="32" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_PreCommencementTenderOffer" xlink:label="loc_deiPreCommencementTenderOffer_50" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_deiPreCommencementTenderOffer_50" xlink:type="arc" order="33" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_PreCommencementIssuerTenderOffer" xlink:label="loc_deiPreCommencementIssuerTenderOffer_50" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_deiPreCommencementIssuerTenderOffer_50" xlink:type="arc" order="34" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_Security12bTitle" xlink:label="loc_deiSecurity12bTitle_50" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_deiSecurity12bTitle_50" xlink:type="arc" order="35" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_NoTradingSymbolFlag" xlink:label="loc_deiNoTradingSymbolFlag_50" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_deiNoTradingSymbolFlag_50" xlink:type="arc" order="36" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_TradingSymbol" xlink:label="loc_deiTradingSymbol_50" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_deiTradingSymbol_50" xlink:type="arc" order="37" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_SecurityExchangeName" xlink:label="loc_deiSecurityExchangeName_50" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_deiSecurityExchangeName_50" xlink:type="arc" order="38" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_Security12gTitle" xlink:label="loc_deiSecurity12gTitle_50" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_deiSecurity12gTitle_50" xlink:type="arc" order="39" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_SecurityReportingObligation" xlink:label="loc_deiSecurityReportingObligation_50" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_deiSecurityReportingObligation_50" xlink:type="arc" order="40" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_AnnualInformationForm" xlink:label="loc_deiAnnualInformationForm_50" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_deiAnnualInformationForm_50" xlink:type="arc" order="41" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_AuditedAnnualFinancialStatements" xlink:label="loc_deiAuditedAnnualFinancialStatements_50" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_deiAuditedAnnualFinancialStatements_50" xlink:type="arc" order="42" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityWellKnownSeasonedIssuer" xlink:label="loc_deiEntityWellKnownSeasonedIssuer_50" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_deiEntityWellKnownSeasonedIssuer_50" xlink:type="arc" order="43" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityVoluntaryFilers" xlink:label="loc_deiEntityVoluntaryFilers_50" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_deiEntityVoluntaryFilers_50" xlink:type="arc" order="44" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityCurrentReportingStatus" xlink:label="loc_deiEntityCurrentReportingStatus_50" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_deiEntityCurrentReportingStatus_50" xlink:type="arc" order="45" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityInteractiveDataCurrent" xlink:label="loc_deiEntityInteractiveDataCurrent_50" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_deiEntityInteractiveDataCurrent_50" xlink:type="arc" order="46" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityFilerCategory" xlink:label="loc_deiEntityFilerCategory_50" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_deiEntityFilerCategory_50" xlink:type="arc" order="47" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntitySmallBusiness" xlink:label="loc_deiEntitySmallBusiness_50" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_deiEntitySmallBusiness_50" xlink:type="arc" order="48" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityEmergingGrowthCompany" xlink:label="loc_deiEntityEmergingGrowthCompany_50" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_deiEntityEmergingGrowthCompany_50" xlink:type="arc" order="49" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityExTransitionPeriod" xlink:label="loc_deiEntityExTransitionPeriod_50" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_deiEntityExTransitionPeriod_50" xlink:type="arc" order="50" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_DocumentAccountingStandard" xlink:label="loc_deiDocumentAccountingStandard_50" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_deiDocumentAccountingStandard_50" xlink:type="arc" order="51" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_OtherReportingStandardItemNumber" xlink:label="loc_deiOtherReportingStandardItemNumber_50" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_deiOtherReportingStandardItemNumber_50" xlink:type="arc" order="52" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityShellCompany" xlink:label="loc_deiEntityShellCompany_50" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_deiEntityShellCompany_50" xlink:type="arc" order="53" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityPublicFloat" xlink:label="loc_deiEntityPublicFloat_50" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_deiEntityPublicFloat_50" xlink:type="arc" order="54" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityBankruptcyProceedingsReportingCurrent" xlink:label="loc_deiEntityBankruptcyProceedingsReportingCurrent_50" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_deiEntityBankruptcyProceedingsReportingCurrent_50" xlink:type="arc" order="55" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityCommonStockSharesOutstanding" xlink:label="loc_deiEntityCommonStockSharesOutstanding_50" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_deiEntityCommonStockSharesOutstanding_50" xlink:type="arc" order="56" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_DocumentsIncorporatedByReferenceTextBlock" xlink:label="loc_deiDocumentsIncorporatedByReferenceTextBlock_50" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_deiDocumentsIncorporatedByReferenceTextBlock_50" xlink:type="arc" order="57" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityListingParValuePerShare" xlink:label="loc_deiEntityListingParValuePerShare_50" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_deiEntityListingParValuePerShare_50" xlink:type="arc" order="58" />
    </link:definitionLink>
    <link:definitionLink xlink:type="extended" xlink:role="http://protagenic.com/role/BalanceSheets" xlink:title="00000002 - Statement - Consolidated Balance Sheets">
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementLineItems" xlink:label="loc_us-gaapStatementLineItems" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementTable" xlink:label="loc_us-gaapStatementTable" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xbrldt:contextElement="segment" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapStatementTable" xlink:type="arc" order="10" xbrldt:closed="true" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementClassOfStockAxis" xlink:label="loc_us-gaapStatementClassOfStockAxis" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaapStatementTable" xlink:to="loc_us-gaapStatementClassOfStockAxis" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfStockDomain" xlink:label="loc_us-gaapClassOfStockDomain" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaapStatementClassOfStockAxis" xlink:to="loc_us-gaapClassOfStockDomain" xlink:type="arc" order="20" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfStockDomain" xlink:label="loc_us-gaapClassOfStockDomain_10" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xbrldt:contextElement="segment" xlink:from="loc_us-gaapStatementClassOfStockAxis" xlink:to="loc_us-gaapClassOfStockDomain_10" xlink:type="arc" order="30" />
      <link:loc xlink:type="locator" xlink:href="ptix-20260630.xsd#PTIX_SeriesAConvertiblePreferredStockMember" xlink:label="loc_PTIXSeriesAConvertiblePreferredStockMember_20" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapClassOfStockDomain" xlink:to="loc_PTIXSeriesAConvertiblePreferredStockMember_20" xlink:type="arc" order="21" />
      <link:loc xlink:type="locator" xlink:href="ptix-20260630.xsd#PTIX_SeriesBConvertiblePreferredStockMember" xlink:label="loc_PTIXSeriesBConvertiblePreferredStockMember_40" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapClassOfStockDomain" xlink:to="loc_PTIXSeriesBConvertiblePreferredStockMember_40" xlink:type="arc" order="41" />
      <link:loc xlink:type="locator" xlink:href="ptix-20260630.xsd#PTIX_SeriesCConvertiblePreferredStockMember" xlink:label="loc_PTIXSeriesCConvertiblePreferredStockMember_60" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapClassOfStockDomain" xlink:to="loc_PTIXSeriesCConvertiblePreferredStockMember_60" xlink:type="arc" order="61" />
      <link:loc xlink:type="locator" xlink:href="ptix-20260630.xsd#PTIX_SeriesC1ConvertiblePreferredStockMember" xlink:label="loc_PTIXSeriesC1ConvertiblePreferredStockMember_80" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapClassOfStockDomain" xlink:to="loc_PTIXSeriesC1ConvertiblePreferredStockMember_80" xlink:type="arc" order="81" />
      <link:loc xlink:type="locator" xlink:href="ptix-20260630.xsd#PTIX_SeriesDConvertiblePreferredStockMember" xlink:label="loc_PTIXSeriesDConvertiblePreferredStockMember_100" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapClassOfStockDomain" xlink:to="loc_PTIXSeriesDConvertiblePreferredStockMember_100" xlink:type="arc" order="101" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AssetsAbstract" xlink:label="loc_us-gaapAssetsAbstract_130" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapAssetsAbstract_130" xlink:type="arc" order="0" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AssetsCurrentAbstract" xlink:label="loc_us-gaapAssetsCurrentAbstract_130" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapAssetsAbstract_130" xlink:to="loc_us-gaapAssetsCurrentAbstract_130" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="ptix-20260630.xsd#PTIX_CashInBank" xlink:label="loc_PTIXCashInBank_130" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapAssetsCurrentAbstract_130" xlink:to="loc_PTIXCashInBank_130" xlink:type="arc" order="2" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_PrepaidExpenseCurrent" xlink:label="loc_us-gaapPrepaidExpenseCurrent_130" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapAssetsCurrentAbstract_130" xlink:to="loc_us-gaapPrepaidExpenseCurrent_130" xlink:type="arc" order="3" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AssetsCurrent" xlink:label="loc_us-gaapAssetsCurrent_130" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapAssetsCurrentAbstract_130" xlink:to="loc_us-gaapAssetsCurrent_130" xlink:type="arc" order="4" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AssetsNoncurrentAbstract" xlink:label="loc_us-gaapAssetsNoncurrentAbstract_130" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapAssetsAbstract_130" xlink:to="loc_us-gaapAssetsNoncurrentAbstract_130" xlink:type="arc" order="5" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_PropertyPlantAndEquipmentNet" xlink:label="loc_us-gaapPropertyPlantAndEquipmentNet_130" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapAssetsNoncurrentAbstract_130" xlink:to="loc_us-gaapPropertyPlantAndEquipmentNet_130" xlink:type="arc" order="6" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_IntangibleAssetsNetExcludingGoodwill" xlink:label="loc_us-gaapIntangibleAssetsNetExcludingGoodwill_130" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapAssetsNoncurrentAbstract_130" xlink:to="loc_us-gaapIntangibleAssetsNetExcludingGoodwill_130" xlink:type="arc" order="7" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AssetsNoncurrent" xlink:label="loc_us-gaapAssetsNoncurrent_130" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapAssetsNoncurrentAbstract_130" xlink:to="loc_us-gaapAssetsNoncurrent_130" xlink:type="arc" order="8" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_Assets" xlink:label="loc_us-gaapAssets_130" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapAssetsAbstract_130" xlink:to="loc_us-gaapAssets_130" xlink:type="arc" order="9" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_LiabilitiesAndStockholdersEquityAbstract" xlink:label="loc_us-gaapLiabilitiesAndStockholdersEquityAbstract_130" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapLiabilitiesAndStockholdersEquityAbstract_130" xlink:type="arc" order="10" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_LiabilitiesAbstract" xlink:label="loc_us-gaapLiabilitiesAbstract_130" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapLiabilitiesAbstract_130" xlink:type="arc" order="11" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_LiabilitiesCurrentAbstract" xlink:label="loc_us-gaapLiabilitiesCurrentAbstract_130" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapLiabilitiesAbstract_130" xlink:to="loc_us-gaapLiabilitiesCurrentAbstract_130" xlink:type="arc" order="12" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AccountsPayableCurrent" xlink:label="loc_us-gaapAccountsPayableCurrent_130" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapLiabilitiesCurrentAbstract_130" xlink:to="loc_us-gaapAccountsPayableCurrent_130" xlink:type="arc" order="13" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AccruedLiabilitiesCurrent" xlink:label="loc_us-gaapAccruedLiabilitiesCurrent_130" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapLiabilitiesCurrentAbstract_130" xlink:to="loc_us-gaapAccruedLiabilitiesCurrent_130" xlink:type="arc" order="14" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_LiabilitiesCurrent" xlink:label="loc_us-gaapLiabilitiesCurrent_130" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapLiabilitiesCurrentAbstract_130" xlink:to="loc_us-gaapLiabilitiesCurrent_130" xlink:type="arc" order="15" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_Liabilities" xlink:label="loc_us-gaapLiabilities_130" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapLiabilitiesAbstract_130" xlink:to="loc_us-gaapLiabilities_130" xlink:type="arc" order="16" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_CommitmentsAndContingencies" xlink:label="loc_us-gaapCommitmentsAndContingencies_130" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapLiabilitiesAbstract_130" xlink:to="loc_us-gaapCommitmentsAndContingencies_130" xlink:type="arc" order="17" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StockholdersEquityAbstract" xlink:label="loc_us-gaapStockholdersEquityAbstract_130" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapLiabilitiesAbstract_130" xlink:to="loc_us-gaapStockholdersEquityAbstract_130" xlink:type="arc" order="18" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_PreferredStockValue" xlink:label="loc_us-gaapPreferredStockValue_130" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStockholdersEquityAbstract_130" xlink:to="loc_us-gaapPreferredStockValue_130" xlink:type="arc" order="19" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_CommonStockValue" xlink:label="loc_us-gaapCommonStockValue_130" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStockholdersEquityAbstract_130" xlink:to="loc_us-gaapCommonStockValue_130" xlink:type="arc" order="20" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AdditionalPaidInCapital" xlink:label="loc_us-gaapAdditionalPaidInCapital_130" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStockholdersEquityAbstract_130" xlink:to="loc_us-gaapAdditionalPaidInCapital_130" xlink:type="arc" order="21" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_RetainedEarningsAccumulatedDeficit" xlink:label="loc_us-gaapRetainedEarningsAccumulatedDeficit_130" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStockholdersEquityAbstract_130" xlink:to="loc_us-gaapRetainedEarningsAccumulatedDeficit_130" xlink:type="arc" order="22" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AccumulatedOtherComprehensiveIncomeLossForeignCurrencyTranslationAdjustmentNetOfTax" xlink:label="loc_us-gaapAccumulatedOtherComprehensiveIncomeLossForeignCurrencyTranslationAdjustmentNetOfTax_130" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStockholdersEquityAbstract_130" xlink:to="loc_us-gaapAccumulatedOtherComprehensiveIncomeLossForeignCurrencyTranslationAdjustmentNetOfTax_130" xlink:type="arc" order="23" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StockholdersEquity" xlink:label="loc_us-gaapStockholdersEquity_130" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStockholdersEquityAbstract_130" xlink:to="loc_us-gaapStockholdersEquity_130" xlink:type="arc" order="24" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_LiabilitiesAndStockholdersEquity" xlink:label="loc_us-gaapLiabilitiesAndStockholdersEquity_130" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapLiabilitiesAbstract_130" xlink:to="loc_us-gaapLiabilitiesAndStockholdersEquity_130" xlink:type="arc" order="25" />
    </link:definitionLink>
    <link:definitionLink xlink:type="extended" xlink:role="http://protagenic.com/role/BalanceSheetsParenthetical" xlink:title="00000003 - Statement - Consolidated Balance Sheets (Parenthetical)">
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementLineItems" xlink:label="loc_us-gaapStatementLineItems" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementTable" xlink:label="loc_us-gaapStatementTable" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xbrldt:contextElement="segment" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapStatementTable" xlink:type="arc" order="10" xbrldt:closed="true" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementClassOfStockAxis" xlink:label="loc_us-gaapStatementClassOfStockAxis" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaapStatementTable" xlink:to="loc_us-gaapStatementClassOfStockAxis" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfStockDomain" xlink:label="loc_us-gaapClassOfStockDomain" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaapStatementClassOfStockAxis" xlink:to="loc_us-gaapClassOfStockDomain" xlink:type="arc" order="20" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfStockDomain" xlink:label="loc_us-gaapClassOfStockDomain_10" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xbrldt:contextElement="segment" xlink:from="loc_us-gaapStatementClassOfStockAxis" xlink:to="loc_us-gaapClassOfStockDomain_10" xlink:type="arc" order="30" />
      <link:loc xlink:type="locator" xlink:href="ptix-20260630.xsd#PTIX_SeriesAConvertiblePreferredStockMember" xlink:label="loc_PTIXSeriesAConvertiblePreferredStockMember_20" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapClassOfStockDomain" xlink:to="loc_PTIXSeriesAConvertiblePreferredStockMember_20" xlink:type="arc" order="21" />
      <link:loc xlink:type="locator" xlink:href="ptix-20260630.xsd#PTIX_SeriesBConvertiblePreferredStockMember" xlink:label="loc_PTIXSeriesBConvertiblePreferredStockMember_40" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapClassOfStockDomain" xlink:to="loc_PTIXSeriesBConvertiblePreferredStockMember_40" xlink:type="arc" order="41" />
      <link:loc xlink:type="locator" xlink:href="ptix-20260630.xsd#PTIX_SeriesCConvertiblePreferredStockMember" xlink:label="loc_PTIXSeriesCConvertiblePreferredStockMember_60" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapClassOfStockDomain" xlink:to="loc_PTIXSeriesCConvertiblePreferredStockMember_60" xlink:type="arc" order="61" />
      <link:loc xlink:type="locator" xlink:href="ptix-20260630.xsd#PTIX_SeriesC1ConvertiblePreferredStockMember" xlink:label="loc_PTIXSeriesC1ConvertiblePreferredStockMember_80" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapClassOfStockDomain" xlink:to="loc_PTIXSeriesC1ConvertiblePreferredStockMember_80" xlink:type="arc" order="81" />
      <link:loc xlink:type="locator" xlink:href="ptix-20260630.xsd#PTIX_SeriesDConvertiblePreferredStockMember" xlink:label="loc_PTIXSeriesDConvertiblePreferredStockMember_100" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapClassOfStockDomain" xlink:to="loc_PTIXSeriesDConvertiblePreferredStockMember_100" xlink:type="arc" order="101" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_PreferredStockParOrStatedValuePerShare" xlink:label="loc_us-gaapPreferredStockParOrStatedValuePerShare_130" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapPreferredStockParOrStatedValuePerShare_130" xlink:type="arc" order="0" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_PreferredStockSharesAuthorized" xlink:label="loc_us-gaapPreferredStockSharesAuthorized_130" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapPreferredStockSharesAuthorized_130" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_PreferredStockSharesIssued" xlink:label="loc_us-gaapPreferredStockSharesIssued_130" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapPreferredStockSharesIssued_130" xlink:type="arc" order="2" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_PreferredStockSharesOutstanding" xlink:label="loc_us-gaapPreferredStockSharesOutstanding_130" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapPreferredStockSharesOutstanding_130" xlink:type="arc" order="3" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_CommonStockParOrStatedValuePerShare" xlink:label="loc_us-gaapCommonStockParOrStatedValuePerShare_130" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapCommonStockParOrStatedValuePerShare_130" xlink:type="arc" order="4" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_CommonStockSharesAuthorized" xlink:label="loc_us-gaapCommonStockSharesAuthorized_130" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapCommonStockSharesAuthorized_130" xlink:type="arc" order="5" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_CommonStockSharesIssued" xlink:label="loc_us-gaapCommonStockSharesIssued_130" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapCommonStockSharesIssued_130" xlink:type="arc" order="6" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_CommonStockSharesOutstanding" xlink:label="loc_us-gaapCommonStockSharesOutstanding_130" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapCommonStockSharesOutstanding_130" xlink:type="arc" order="7" />
    </link:definitionLink>
    <link:definitionLink xlink:type="extended" xlink:role="http://protagenic.com/role/StatementsOfOperationsAndComprehensiveIncomeLoss" xlink:title="00000004 - Statement - Consolidated Statements of Operations and Comprehensive Income (Loss) (Unaudited)" />
    <link:definitionLink xlink:type="extended" xlink:role="http://protagenic.com/role/StatementsOfChangeInStockholdersEquity" xlink:title="00000005 - Statement - Consolidated Statements of Change in Stockholders&apos; Equity (Unaudited)">
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementLineItems" xlink:label="loc_us-gaapStatementLineItems" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementTable" xlink:label="loc_us-gaapStatementTable" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xbrldt:contextElement="segment" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapStatementTable" xlink:type="arc" order="10" xbrldt:closed="true" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementEquityComponentsAxis" xlink:label="loc_us-gaapStatementEquityComponentsAxis" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaapStatementTable" xlink:to="loc_us-gaapStatementEquityComponentsAxis" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_EquityComponentDomain" xlink:label="loc_us-gaapEquityComponentDomain" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaapStatementEquityComponentsAxis" xlink:to="loc_us-gaapEquityComponentDomain" xlink:type="arc" order="20" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_EquityComponentDomain" xlink:label="loc_us-gaapEquityComponentDomain_10" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xbrldt:contextElement="segment" xlink:from="loc_us-gaapStatementEquityComponentsAxis" xlink:to="loc_us-gaapEquityComponentDomain_10" xlink:type="arc" order="30" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_PreferredStockMember" xlink:label="loc_us-gaapPreferredStockMember_0" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapEquityComponentDomain" xlink:to="loc_us-gaapPreferredStockMember_0" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_CommonStockMember" xlink:label="loc_us-gaapCommonStockMember_50" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapEquityComponentDomain" xlink:to="loc_us-gaapCommonStockMember_50" xlink:type="arc" order="51" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AdditionalPaidInCapitalMember" xlink:label="loc_us-gaapAdditionalPaidInCapitalMember_60" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapEquityComponentDomain" xlink:to="loc_us-gaapAdditionalPaidInCapitalMember_60" xlink:type="arc" order="61" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AccumulatedOtherComprehensiveIncomeMember" xlink:label="loc_us-gaapAccumulatedOtherComprehensiveIncomeMember_70" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapEquityComponentDomain" xlink:to="loc_us-gaapAccumulatedOtherComprehensiveIncomeMember_70" xlink:type="arc" order="71" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_RetainedEarningsMember" xlink:label="loc_us-gaapRetainedEarningsMember_80" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapEquityComponentDomain" xlink:to="loc_us-gaapRetainedEarningsMember_80" xlink:type="arc" order="81" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementClassOfStockAxis" xlink:label="loc_us-gaapStatementClassOfStockAxis" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaapStatementTable" xlink:to="loc_us-gaapStatementClassOfStockAxis" xlink:type="arc" order="2" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfStockDomain" xlink:label="loc_us-gaapClassOfStockDomain" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaapStatementClassOfStockAxis" xlink:to="loc_us-gaapClassOfStockDomain" xlink:type="arc" order="20" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfStockDomain" xlink:label="loc_us-gaapClassOfStockDomain_10" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xbrldt:contextElement="segment" xlink:from="loc_us-gaapStatementClassOfStockAxis" xlink:to="loc_us-gaapClassOfStockDomain_10" xlink:type="arc" order="30" />
      <link:loc xlink:type="locator" xlink:href="ptix-20260630.xsd#PTIX_SeriesAConvertiblePreferredStockMember" xlink:label="loc_PTIXSeriesAConvertiblePreferredStockMember_0" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapClassOfStockDomain" xlink:to="loc_PTIXSeriesAConvertiblePreferredStockMember_0" xlink:type="arc" order="2" />
      <link:loc xlink:type="locator" xlink:href="ptix-20260630.xsd#PTIX_SeriesBConvertiblePreferredStockMember" xlink:label="loc_PTIXSeriesBConvertiblePreferredStockMember_10" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapClassOfStockDomain" xlink:to="loc_PTIXSeriesBConvertiblePreferredStockMember_10" xlink:type="arc" order="12" />
      <link:loc xlink:type="locator" xlink:href="ptix-20260630.xsd#PTIX_SeriesCConvertiblePreferredStockMember" xlink:label="loc_PTIXSeriesCConvertiblePreferredStockMember_20" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapClassOfStockDomain" xlink:to="loc_PTIXSeriesCConvertiblePreferredStockMember_20" xlink:type="arc" order="22" />
      <link:loc xlink:type="locator" xlink:href="ptix-20260630.xsd#PTIX_SeriesC1ConvertiblePreferredStockMember" xlink:label="loc_PTIXSeriesC1ConvertiblePreferredStockMember_30" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapClassOfStockDomain" xlink:to="loc_PTIXSeriesC1ConvertiblePreferredStockMember_30" xlink:type="arc" order="32" />
      <link:loc xlink:type="locator" xlink:href="ptix-20260630.xsd#PTIX_SeriesDConvertiblePreferredStockMember" xlink:label="loc_PTIXSeriesDConvertiblePreferredStockMember_40" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapClassOfStockDomain" xlink:to="loc_PTIXSeriesDConvertiblePreferredStockMember_40" xlink:type="arc" order="42" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StockholdersEquity" xlink:label="loc_us-gaapStockholdersEquity_110" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapStockholdersEquity_110" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SharesOutstanding" xlink:label="loc_us-gaapSharesOutstanding_110" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapSharesOutstanding_110" xlink:type="arc" order="2" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_OtherComprehensiveIncomeForeignCurrencyTransactionAndTranslationGainLossBeforeReclassificationAndTax" xlink:label="loc_us-gaapOtherComprehensiveIncomeForeignCurrencyTransactionAndTranslationGainLossBeforeReclassificationAndTax_110" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapOtherComprehensiveIncomeForeignCurrencyTransactionAndTranslationGainLossBeforeReclassificationAndTax_110" xlink:type="arc" order="3" />
      <link:loc xlink:type="locator" xlink:href="ptix-20260630.xsd#PTIX_StockIssuedDuringPeriodValuePreferredStockToOfficers" xlink:label="loc_PTIXStockIssuedDuringPeriodValuePreferredStockToOfficers_110" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_PTIXStockIssuedDuringPeriodValuePreferredStockToOfficers_110" xlink:type="arc" order="4" />
      <link:loc xlink:type="locator" xlink:href="ptix-20260630.xsd#PTIX_StockIssuedDuringPeriodSharesPreferredStockToOfficers" xlink:label="loc_PTIXStockIssuedDuringPeriodSharesPreferredStockToOfficers_110" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_PTIXStockIssuedDuringPeriodSharesPreferredStockToOfficers_110" xlink:type="arc" order="5" />
      <link:loc xlink:type="locator" xlink:href="ptix-20260630.xsd#PTIX_StockIssuedDuringPeriodValueReverseMerger" xlink:label="loc_PTIXStockIssuedDuringPeriodValueReverseMerger_110" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_PTIXStockIssuedDuringPeriodValueReverseMerger_110" xlink:type="arc" order="6" />
      <link:loc xlink:type="locator" xlink:href="ptix-20260630.xsd#PTIX_StockIssuedDuringPeriodSharesReverseMerger" xlink:label="loc_PTIXStockIssuedDuringPeriodSharesReverseMerger_110" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_PTIXStockIssuedDuringPeriodSharesReverseMerger_110" xlink:type="arc" order="7" />
      <link:loc xlink:type="locator" xlink:href="ptix-20260630.xsd#PTIX_StockIssuedDuringPeriodValueBioPurchase" xlink:label="loc_PTIXStockIssuedDuringPeriodValueBioPurchase_110" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_PTIXStockIssuedDuringPeriodValueBioPurchase_110" xlink:type="arc" order="8" />
      <link:loc xlink:type="locator" xlink:href="ptix-20260630.xsd#PTIX_StockIssuedDuringPeriodSharesBioPurchase" xlink:label="loc_PTIXStockIssuedDuringPeriodSharesBioPurchase_110" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_PTIXStockIssuedDuringPeriodSharesBioPurchase_110" xlink:type="arc" order="9" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StockIssuedDuringPeriodValueNewIssues" xlink:label="loc_us-gaapStockIssuedDuringPeriodValueNewIssues_110" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapStockIssuedDuringPeriodValueNewIssues_110" xlink:type="arc" order="10" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StockIssuedDuringPeriodSharesNewIssues" xlink:label="loc_us-gaapStockIssuedDuringPeriodSharesNewIssues_110" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapStockIssuedDuringPeriodSharesNewIssues_110" xlink:type="arc" order="11" />
      <link:loc xlink:type="locator" xlink:href="ptix-20260630.xsd#PTIX_StockIssuedDuringPeriodValueWarrantExercise" xlink:label="loc_PTIXStockIssuedDuringPeriodValueWarrantExercise_110" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_PTIXStockIssuedDuringPeriodValueWarrantExercise_110" xlink:type="arc" order="12" />
      <link:loc xlink:type="locator" xlink:href="ptix-20260630.xsd#PTIX_StockIssuedDuringPeriodSharesWarrantExercise" xlink:label="loc_PTIXStockIssuedDuringPeriodSharesWarrantExercise_110" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_PTIXStockIssuedDuringPeriodSharesWarrantExercise_110" xlink:type="arc" order="13" />
      <link:loc xlink:type="locator" xlink:href="ptix-20260630.xsd#PTIX_StockIssuedDuringPeriodValueOptionExercise" xlink:label="loc_PTIXStockIssuedDuringPeriodValueOptionExercise_110" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_PTIXStockIssuedDuringPeriodValueOptionExercise_110" xlink:type="arc" order="14" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AdjustmentsToAdditionalPaidInCapitalSharebasedCompensationRequisiteServicePeriodRecognitionValue" xlink:label="loc_us-gaapAdjustmentsToAdditionalPaidInCapitalSharebasedCompensationRequisiteServicePeriodRecognitionValue_110" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapAdjustmentsToAdditionalPaidInCapitalSharebasedCompensationRequisiteServicePeriodRecognitionValue_110" xlink:type="arc" order="15" />
      <link:loc xlink:type="locator" xlink:href="ptix-20260630.xsd#PTIX_RoundingFromReverseSplit" xlink:label="loc_PTIXRoundingFromReverseSplit_110" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_PTIXRoundingFromReverseSplit_110" xlink:type="arc" order="16" />
      <link:loc xlink:type="locator" xlink:href="ptix-20260630.xsd#PTIX_RoundingFromReverseSplitShares" xlink:label="loc_PTIXRoundingFromReverseSplitShares_110" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_PTIXRoundingFromReverseSplitShares_110" xlink:type="arc" order="17" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_NetIncomeLoss" xlink:label="loc_us-gaapNetIncomeLoss_110" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapNetIncomeLoss_110" xlink:type="arc" order="18" />
    </link:definitionLink>
    <link:definitionLink xlink:type="extended" xlink:role="http://protagenic.com/role/StatementsOfCashFlows" xlink:title="00000006 - Statement - Consolidated Statements of Cash Flows (Unaudited)" />
    <link:definitionLink xlink:type="extended" xlink:role="http://protagenic.com/role/OrganizationAndNatureOfBusiness" xlink:title="999007 - Disclosure - ORGANIZATION AND NATURE OF BUSINESS" />
    <link:definitionLink xlink:type="extended" xlink:role="http://protagenic.com/role/LiquidityAndGoingConcern" xlink:title="999008 - Disclosure - LIQUIDITY AND GOING CONCERN" />
    <link:definitionLink xlink:type="extended" xlink:role="http://protagenic.com/role/SummaryOfSignificantAccountingPolicies" xlink:title="999009 - Disclosure - SUMMARY OF SIGNIFICANT ACCOUNTING POLICIES" />
    <link:definitionLink xlink:type="extended" xlink:role="http://protagenic.com/role/ReverseMerger" xlink:title="999010 - Disclosure - REVERSE MERGER" />
    <link:definitionLink xlink:type="extended" xlink:role="http://protagenic.com/role/FixedAssets" xlink:title="999011 - Disclosure - FIXED ASSETS" />
    <link:definitionLink xlink:type="extended" xlink:role="http://protagenic.com/role/IntangibleAssets" xlink:title="999012 - Disclosure - INTANGIBLE ASSETS" />
    <link:definitionLink xlink:type="extended" xlink:role="http://protagenic.com/role/AccountsPayable" xlink:title="999013 - Disclosure - ACCOUNTS PAYABLE" />
    <link:definitionLink xlink:type="extended" xlink:role="http://protagenic.com/role/AccruedLiabilities" xlink:title="999014 - Disclosure - ACCRUED LIABILITIES" />
    <link:definitionLink xlink:type="extended" xlink:role="http://protagenic.com/role/RelatedPartyTransactions" xlink:title="999015 - Disclosure - RELATED PARTY TRANSACTIONS" />
    <link:definitionLink xlink:type="extended" xlink:role="http://protagenic.com/role/StockholdersEquity" xlink:title="999016 - Disclosure - STOCKHOLDERS&#8217; EQUITY" />
    <link:definitionLink xlink:type="extended" xlink:role="http://protagenic.com/role/CollaborativeAgreements" xlink:title="999017 - Disclosure - COLLABORATIVE AGREEMENTS" />
    <link:definitionLink xlink:type="extended" xlink:role="http://protagenic.com/role/CommitmentsAndContingencies" xlink:title="999018 - Disclosure - COMMITMENTS AND CONTINGENCIES" />
    <link:definitionLink xlink:type="extended" xlink:role="http://protagenic.com/role/SegmentReporting" xlink:title="999019 - Disclosure - SEGMENT REPORTING" />
    <link:definitionLink xlink:type="extended" xlink:role="http://protagenic.com/role/SubsequentEvents" xlink:title="999020 - Disclosure - SUBSEQUENT EVENTS" />
    <link:definitionLink xlink:type="extended" xlink:role="http://protagenic.com/role/SummaryOfSignificantAccountingPoliciesPolicies" xlink:title="999021 - Disclosure - SUMMARY OF SIGNIFICANT ACCOUNTING POLICIES (Policies)" />
    <link:definitionLink xlink:type="extended" xlink:role="http://protagenic.com/role/SummaryOfSignificantAccountingPoliciesTables" xlink:title="999022 - Disclosure - SUMMARY OF SIGNIFICANT ACCOUNTING POLICIES (Tables)" />
    <link:definitionLink xlink:type="extended" xlink:role="http://protagenic.com/role/ReverseMergerTables" xlink:title="999023 - Disclosure - REVERSE MERGER (Tables)" />
    <link:definitionLink xlink:type="extended" xlink:role="http://protagenic.com/role/FixedAssetsTables" xlink:title="999024 - Disclosure - FIXED ASSETS (Tables)" />
    <link:definitionLink xlink:type="extended" xlink:role="http://protagenic.com/role/IntangibleAssetsTables" xlink:title="999025 - Disclosure - INTANGIBLE ASSETS (Tables)" />
    <link:definitionLink xlink:type="extended" xlink:role="http://protagenic.com/role/AccountsPayableTables" xlink:title="999026 - Disclosure - ACCOUNTS PAYABLE (Tables)" />
    <link:definitionLink xlink:type="extended" xlink:role="http://protagenic.com/role/AccruedLiabilitiesTables" xlink:title="999027 - Disclosure - ACCRUED LIABILITIES (Tables)" />
    <link:definitionLink xlink:type="extended" xlink:role="http://protagenic.com/role/StockholdersEquityTables" xlink:title="999028 - Disclosure - STOCKHOLDERS&#8217; EQUITY (Tables)" />
    <link:definitionLink xlink:type="extended" xlink:role="http://protagenic.com/role/OrganizationAndNatureOfBusinessDetailsNarrative" xlink:title="999029 - Disclosure - ORGANIZATION AND NATURE OF BUSINESS (Details Narrative)" />
    <link:definitionLink xlink:type="extended" xlink:role="http://protagenic.com/role/LiquidityAndGoingConcernDetailsNarrative" xlink:title="999030 - Disclosure - LIQUIDITY AND GOING CONCERN (Details Narrative)" />
    <link:definitionLink xlink:type="extended" xlink:role="http://protagenic.com/role/ScheduleOfAnti-dilutiveSecuritiesExcludedFromComputationOfEarningsPerShareDetails" xlink:title="999031 - Disclosure - SCHEDULE OF ANTI-DILUTIVE SECURITIES EXCLUDED FROM COMPUTATION OF EARNINGS PER SHARE (Details)">
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareLineItems" xlink:label="loc_us-gaapAntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareLineItems" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ScheduleOfAntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareTable" xlink:label="loc_us-gaapScheduleOfAntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareTable" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xbrldt:contextElement="segment" xlink:from="loc_us-gaapAntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareLineItems" xlink:to="loc_us-gaapScheduleOfAntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareTable" xlink:type="arc" order="10" xbrldt:closed="true" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareByAntidilutiveSecuritiesAxis" xlink:label="loc_us-gaapAntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareByAntidilutiveSecuritiesAxis" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaapScheduleOfAntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareTable" xlink:to="loc_us-gaapAntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareByAntidilutiveSecuritiesAxis" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AntidilutiveSecuritiesNameDomain" xlink:label="loc_us-gaapAntidilutiveSecuritiesNameDomain" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaapAntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareByAntidilutiveSecuritiesAxis" xlink:to="loc_us-gaapAntidilutiveSecuritiesNameDomain" xlink:type="arc" order="20" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AntidilutiveSecuritiesNameDomain" xlink:label="loc_us-gaapAntidilutiveSecuritiesNameDomain_10" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xbrldt:contextElement="segment" xlink:from="loc_us-gaapAntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareByAntidilutiveSecuritiesAxis" xlink:to="loc_us-gaapAntidilutiveSecuritiesNameDomain_10" xlink:type="arc" order="30" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_PreferredStockMember" xlink:label="loc_us-gaapPreferredStockMember_0" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapAntidilutiveSecuritiesNameDomain" xlink:to="loc_us-gaapPreferredStockMember_0" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ConvertibleDebtSecuritiesMember" xlink:label="loc_us-gaapConvertibleDebtSecuritiesMember_20" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapAntidilutiveSecuritiesNameDomain" xlink:to="loc_us-gaapConvertibleDebtSecuritiesMember_20" xlink:type="arc" order="21" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_EmployeeStockOptionMember" xlink:label="loc_us-gaapEmployeeStockOptionMember_40" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapAntidilutiveSecuritiesNameDomain" xlink:to="loc_us-gaapEmployeeStockOptionMember_40" xlink:type="arc" order="41" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_WarrantMember" xlink:label="loc_us-gaapWarrantMember_60" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapAntidilutiveSecuritiesNameDomain" xlink:to="loc_us-gaapWarrantMember_60" xlink:type="arc" order="61" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareAmount" xlink:label="loc_us-gaapAntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareAmount_110" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapAntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareLineItems" xlink:to="loc_us-gaapAntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareAmount_110" xlink:type="arc" order="0" />
    </link:definitionLink>
    <link:definitionLink xlink:type="extended" xlink:role="http://protagenic.com/role/SummaryOfSignificantAccountingPoliciesDetailsNarrative" xlink:title="999032 - Disclosure - SUMMARY OF SIGNIFICANT ACCOUNTING POLICIES (Details Narrative)" />
    <link:definitionLink xlink:type="extended" xlink:role="http://protagenic.com/role/ScheduleOfPurchasePriceAllocationDetails" xlink:title="999033 - Disclosure - SCHEDULE OF PURCHASE PRICE ALLOCATION (Details)" />
    <link:definitionLink xlink:type="extended" xlink:role="http://protagenic.com/role/ScheduleOfBusinessCombinationContingentConsiderationDetails" xlink:title="999034 - Disclosure - SCHEDULE OF BUSINESS COMBINATION CONTINGENT CONSIDERATION (Details)" />
    <link:definitionLink xlink:type="extended" xlink:role="http://protagenic.com/role/ReverseMergerDetailsNarrative" xlink:title="999035 - Disclosure - REVERSE MERGER (Details Narrative)">
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_BusinessAcquisitionLineItems" xlink:label="loc_us-gaapBusinessAcquisitionLineItems" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ScheduleOfBusinessAcquisitionsByAcquisitionTable" xlink:label="loc_us-gaapScheduleOfBusinessAcquisitionsByAcquisitionTable" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xbrldt:contextElement="segment" xlink:from="loc_us-gaapBusinessAcquisitionLineItems" xlink:to="loc_us-gaapScheduleOfBusinessAcquisitionsByAcquisitionTable" xlink:type="arc" order="10" xbrldt:closed="true" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementEquityComponentsAxis" xlink:label="loc_us-gaapStatementEquityComponentsAxis" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaapScheduleOfBusinessAcquisitionsByAcquisitionTable" xlink:to="loc_us-gaapStatementEquityComponentsAxis" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_EquityComponentDomain" xlink:label="loc_us-gaapEquityComponentDomain" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaapStatementEquityComponentsAxis" xlink:to="loc_us-gaapEquityComponentDomain" xlink:type="arc" order="20" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_EquityComponentDomain" xlink:label="loc_us-gaapEquityComponentDomain_10" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xbrldt:contextElement="segment" xlink:from="loc_us-gaapStatementEquityComponentsAxis" xlink:to="loc_us-gaapEquityComponentDomain_10" xlink:type="arc" order="30" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_CommonStockMember" xlink:label="loc_us-gaapCommonStockMember_0" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapEquityComponentDomain" xlink:to="loc_us-gaapCommonStockMember_0" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_BusinessAcquisitionAxis" xlink:label="loc_us-gaapBusinessAcquisitionAxis" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaapScheduleOfBusinessAcquisitionsByAcquisitionTable" xlink:to="loc_us-gaapBusinessAcquisitionAxis" xlink:type="arc" order="2" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_BusinessAcquisitionAcquireeDomain" xlink:label="loc_us-gaapBusinessAcquisitionAcquireeDomain" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaapBusinessAcquisitionAxis" xlink:to="loc_us-gaapBusinessAcquisitionAcquireeDomain" xlink:type="arc" order="20" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_BusinessAcquisitionAcquireeDomain" xlink:label="loc_us-gaapBusinessAcquisitionAcquireeDomain_10" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xbrldt:contextElement="segment" xlink:from="loc_us-gaapBusinessAcquisitionAxis" xlink:to="loc_us-gaapBusinessAcquisitionAcquireeDomain_10" xlink:type="arc" order="30" />
      <link:loc xlink:type="locator" xlink:href="ptix-20260630.xsd#PTIX_PhytanixBioIncMember" xlink:label="loc_PTIXPhytanixBioIncMember_0" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapBusinessAcquisitionAcquireeDomain" xlink:to="loc_PTIXPhytanixBioIncMember_0" xlink:type="arc" order="2" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementClassOfStockAxis" xlink:label="loc_us-gaapStatementClassOfStockAxis" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaapScheduleOfBusinessAcquisitionsByAcquisitionTable" xlink:to="loc_us-gaapStatementClassOfStockAxis" xlink:type="arc" order="3" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfStockDomain" xlink:label="loc_us-gaapClassOfStockDomain" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaapStatementClassOfStockAxis" xlink:to="loc_us-gaapClassOfStockDomain" xlink:type="arc" order="20" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfStockDomain" xlink:label="loc_us-gaapClassOfStockDomain_10" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xbrldt:contextElement="segment" xlink:from="loc_us-gaapStatementClassOfStockAxis" xlink:to="loc_us-gaapClassOfStockDomain_10" xlink:type="arc" order="30" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SeriesCPreferredStockMember" xlink:label="loc_us-gaapSeriesCPreferredStockMember_20" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapClassOfStockDomain" xlink:to="loc_us-gaapSeriesCPreferredStockMember_20" xlink:type="arc" order="21" />
      <link:loc xlink:type="locator" xlink:href="ptix-20260630.xsd#PTIX_SeriesCOnePreferredStockMember" xlink:label="loc_PTIXSeriesCOnePreferredStockMember_40" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapClassOfStockDomain" xlink:to="loc_PTIXSeriesCOnePreferredStockMember_40" xlink:type="arc" order="41" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SeriesDPreferredStockMember" xlink:label="loc_us-gaapSeriesDPreferredStockMember_60" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapClassOfStockDomain" xlink:to="loc_us-gaapSeriesDPreferredStockMember_60" xlink:type="arc" order="61" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_ScheduleOfEquityMethodInvestmentEquityMethodInvesteeNameAxis" xlink:label="loc_srtScheduleOfEquityMethodInvestmentEquityMethodInvesteeNameAxis" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaapScheduleOfBusinessAcquisitionsByAcquisitionTable" xlink:to="loc_srtScheduleOfEquityMethodInvestmentEquityMethodInvesteeNameAxis" xlink:type="arc" order="4" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_EquityMethodInvesteeNameDomain" xlink:label="loc_srtEquityMethodInvesteeNameDomain" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_srtScheduleOfEquityMethodInvestmentEquityMethodInvesteeNameAxis" xlink:to="loc_srtEquityMethodInvesteeNameDomain" xlink:type="arc" order="20" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_EquityMethodInvesteeNameDomain" xlink:label="loc_srtEquityMethodInvesteeNameDomain_10" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xbrldt:contextElement="segment" xlink:from="loc_srtScheduleOfEquityMethodInvestmentEquityMethodInvesteeNameAxis" xlink:to="loc_srtEquityMethodInvesteeNameDomain_10" xlink:type="arc" order="30" />
      <link:loc xlink:type="locator" xlink:href="ptix-20260630.xsd#PTIX_PhytanixBioIncMember" xlink:label="loc_PTIXPhytanixBioIncMember_90" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srtEquityMethodInvesteeNameDomain" xlink:to="loc_PTIXPhytanixBioIncMember_90" xlink:type="arc" order="91" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_TypeOfArrangementAxis" xlink:label="loc_us-gaapTypeOfArrangementAxis" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaapScheduleOfBusinessAcquisitionsByAcquisitionTable" xlink:to="loc_us-gaapTypeOfArrangementAxis" xlink:type="arc" order="5" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:label="loc_us-gaapArrangementsAndNonarrangementTransactionsMember" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaapTypeOfArrangementAxis" xlink:to="loc_us-gaapArrangementsAndNonarrangementTransactionsMember" xlink:type="arc" order="20" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:label="loc_us-gaapArrangementsAndNonarrangementTransactionsMember_10" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xbrldt:contextElement="segment" xlink:from="loc_us-gaapTypeOfArrangementAxis" xlink:to="loc_us-gaapArrangementsAndNonarrangementTransactionsMember_10" xlink:type="arc" order="30" />
      <link:loc xlink:type="locator" xlink:href="ptix-20260630.xsd#PTIX_UnwindAgreementMember" xlink:label="loc_PTIXUnwindAgreementMember_100" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapArrangementsAndNonarrangementTransactionsMember" xlink:to="loc_PTIXUnwindAgreementMember_100" xlink:type="arc" order="101" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StockIssuedDuringPeriodSharesNewIssues" xlink:label="loc_us-gaapStockIssuedDuringPeriodSharesNewIssues_130" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapBusinessAcquisitionLineItems" xlink:to="loc_us-gaapStockIssuedDuringPeriodSharesNewIssues_130" xlink:type="arc" order="0" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_CommonStockParOrStatedValuePerShare" xlink:label="loc_us-gaapCommonStockParOrStatedValuePerShare_130" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapBusinessAcquisitionLineItems" xlink:to="loc_us-gaapCommonStockParOrStatedValuePerShare_130" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_BusinessCombinationStepAcquisitionEquityInterestInAcquireeIncludingSubsequentAcquisitionPercentage" xlink:label="loc_us-gaapBusinessCombinationStepAcquisitionEquityInterestInAcquireeIncludingSubsequentAcquisitionPercentage_130" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapBusinessAcquisitionLineItems" xlink:to="loc_us-gaapBusinessCombinationStepAcquisitionEquityInterestInAcquireeIncludingSubsequentAcquisitionPercentage_130" xlink:type="arc" order="2" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_PreferredStockParOrStatedValuePerShare" xlink:label="loc_us-gaapPreferredStockParOrStatedValuePerShare_130" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapBusinessAcquisitionLineItems" xlink:to="loc_us-gaapPreferredStockParOrStatedValuePerShare_130" xlink:type="arc" order="3" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfWarrantOrRightOutstanding" xlink:label="loc_us-gaapClassOfWarrantOrRightOutstanding_130" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapBusinessAcquisitionLineItems" xlink:to="loc_us-gaapClassOfWarrantOrRightOutstanding_130" xlink:type="arc" order="4" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ImpairmentOfLongLivedAssetsToBeDisposedOf" xlink:label="loc_us-gaapImpairmentOfLongLivedAssetsToBeDisposedOf_130" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapBusinessAcquisitionLineItems" xlink:to="loc_us-gaapImpairmentOfLongLivedAssetsToBeDisposedOf_130" xlink:type="arc" order="5" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_EquityMethodInvestmentOwnershipPercentage" xlink:label="loc_us-gaapEquityMethodInvestmentOwnershipPercentage_130" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapBusinessAcquisitionLineItems" xlink:to="loc_us-gaapEquityMethodInvestmentOwnershipPercentage_130" xlink:type="arc" order="6" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DebtSecuritiesCurrent" xlink:label="loc_us-gaapDebtSecuritiesCurrent_130" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapBusinessAcquisitionLineItems" xlink:to="loc_us-gaapDebtSecuritiesCurrent_130" xlink:type="arc" order="7" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DebtSecuritiesNoncurrent" xlink:label="loc_us-gaapDebtSecuritiesNoncurrent_130" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapBusinessAcquisitionLineItems" xlink:to="loc_us-gaapDebtSecuritiesNoncurrent_130" xlink:type="arc" order="8" />
      <link:loc xlink:type="locator" xlink:href="ptix-20260630.xsd#PTIX_BusinessCombinationSeparatelyRecognizedTransactionsNetGainAndLosses" xlink:label="loc_PTIXBusinessCombinationSeparatelyRecognizedTransactionsNetGainAndLosses_130" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapBusinessAcquisitionLineItems" xlink:to="loc_PTIXBusinessCombinationSeparatelyRecognizedTransactionsNetGainAndLosses_130" xlink:type="arc" order="9" />
    </link:definitionLink>
    <link:definitionLink xlink:type="extended" xlink:role="http://protagenic.com/role/ScheduleOfFixedAssetsDetails" xlink:title="999036 - Disclosure - SCHEDULE OF FIXED ASSETS (Details)" />
    <link:definitionLink xlink:type="extended" xlink:role="http://protagenic.com/role/FixedAssetsDetailsNarrative" xlink:title="999037 - Disclosure - FIXED ASSETS (Details Narrative)" />
    <link:definitionLink xlink:type="extended" xlink:role="http://protagenic.com/role/ScheduleOfIntangibleAssetsDetails" xlink:title="999038 - Disclosure - SCHEDULE OF INTANGIBLE ASSETS (Details)">
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_FiniteLivedIntangibleAssetsLineItems" xlink:label="loc_us-gaapFiniteLivedIntangibleAssetsLineItems" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ScheduleOfFiniteLivedIntangibleAssetsTable" xlink:label="loc_us-gaapScheduleOfFiniteLivedIntangibleAssetsTable" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xbrldt:contextElement="segment" xlink:from="loc_us-gaapFiniteLivedIntangibleAssetsLineItems" xlink:to="loc_us-gaapScheduleOfFiniteLivedIntangibleAssetsTable" xlink:type="arc" order="10" xbrldt:closed="true" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_FiniteLivedIntangibleAssetsByMajorClassAxis" xlink:label="loc_us-gaapFiniteLivedIntangibleAssetsByMajorClassAxis" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaapScheduleOfFiniteLivedIntangibleAssetsTable" xlink:to="loc_us-gaapFiniteLivedIntangibleAssetsByMajorClassAxis" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_FiniteLivedIntangibleAssetsMajorClassNameDomain" xlink:label="loc_us-gaapFiniteLivedIntangibleAssetsMajorClassNameDomain" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaapFiniteLivedIntangibleAssetsByMajorClassAxis" xlink:to="loc_us-gaapFiniteLivedIntangibleAssetsMajorClassNameDomain" xlink:type="arc" order="20" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_FiniteLivedIntangibleAssetsMajorClassNameDomain" xlink:label="loc_us-gaapFiniteLivedIntangibleAssetsMajorClassNameDomain_10" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xbrldt:contextElement="segment" xlink:from="loc_us-gaapFiniteLivedIntangibleAssetsByMajorClassAxis" xlink:to="loc_us-gaapFiniteLivedIntangibleAssetsMajorClassNameDomain_10" xlink:type="arc" order="30" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_InProcessResearchAndDevelopmentMember" xlink:label="loc_us-gaapInProcessResearchAndDevelopmentMember_0" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapFiniteLivedIntangibleAssetsMajorClassNameDomain" xlink:to="loc_us-gaapInProcessResearchAndDevelopmentMember_0" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="ptix-20260630.xsd#PTIX_AssembledWorkforceMember" xlink:label="loc_PTIXAssembledWorkforceMember_20" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapFiniteLivedIntangibleAssetsMajorClassNameDomain" xlink:to="loc_PTIXAssembledWorkforceMember_20" xlink:type="arc" order="21" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_FiniteLivedIntangibleAssetsGross" xlink:label="loc_us-gaapFiniteLivedIntangibleAssetsGross_70" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapFiniteLivedIntangibleAssetsLineItems" xlink:to="loc_us-gaapFiniteLivedIntangibleAssetsGross_70" xlink:type="arc" order="0" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_FiniteLivedIntangibleAssetsAccumulatedAmortization" xlink:label="loc_us-gaapFiniteLivedIntangibleAssetsAccumulatedAmortization_70" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapFiniteLivedIntangibleAssetsLineItems" xlink:to="loc_us-gaapFiniteLivedIntangibleAssetsAccumulatedAmortization_70" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_FiniteLivedIntangibleAssetsNet" xlink:label="loc_us-gaapFiniteLivedIntangibleAssetsNet_70" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapFiniteLivedIntangibleAssetsLineItems" xlink:to="loc_us-gaapFiniteLivedIntangibleAssetsNet_70" xlink:type="arc" order="2" />
    </link:definitionLink>
    <link:definitionLink xlink:type="extended" xlink:role="http://protagenic.com/role/IntangibleAssetsDetailsNarrative" xlink:title="999039 - Disclosure - INTANGIBLE ASSETS (Details Narrative)" />
    <link:definitionLink xlink:type="extended" xlink:role="http://protagenic.com/role/ScheduleOfAccountsPayableDetails" xlink:title="999040 - Disclosure - SCHEDULE OF ACCOUNTS PAYABLE (Details)" />
    <link:definitionLink xlink:type="extended" xlink:role="http://protagenic.com/role/ScheduleOfAccruedLiabilitiesDetails" xlink:title="999041 - Disclosure - SCHEDULE OF ACCRUED LIABILITIES (Details)" />
    <link:definitionLink xlink:type="extended" xlink:role="http://protagenic.com/role/RelatedPartyTransactionsDetailsNarrative" xlink:title="999042 - Disclosure - RELATED PARTY TRANSACTIONS (Details Narrative)" />
    <link:definitionLink xlink:type="extended" xlink:role="http://protagenic.com/role/ScheduleOfFairValueOfEachOptionGrantedDetails" xlink:title="999043 - Disclosure - SCHEDULE OF FAIR VALUE OF EACH OPTION GRANTED (Details)" />
    <link:definitionLink xlink:type="extended" xlink:role="http://protagenic.com/role/ScheduleOfShare-basedCompensationStockOptionsActivityDetails" xlink:title="999044 - Disclosure - SCHEDULE OF SHARE-BASED COMPENSATION, STOCK OPTIONS, ACTIVITY (Details)">
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:label="loc_us-gaapShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable" xlink:label="loc_us-gaapScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xbrldt:contextElement="segment" xlink:from="loc_us-gaapShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="loc_us-gaapScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable" xlink:type="arc" order="10" xbrldt:closed="true" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AwardTypeAxis" xlink:label="loc_us-gaapAwardTypeAxis" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaapScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable" xlink:to="loc_us-gaapAwardTypeAxis" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:label="loc_us-gaapShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaapAwardTypeAxis" xlink:to="loc_us-gaapShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:type="arc" order="20" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:label="loc_us-gaapShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_10" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xbrldt:contextElement="segment" xlink:from="loc_us-gaapAwardTypeAxis" xlink:to="loc_us-gaapShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_10" xlink:type="arc" order="30" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_EmployeeStockOptionMember" xlink:label="loc_us-gaapEmployeeStockOptionMember_0" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:to="loc_us-gaapEmployeeStockOptionMember_0" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingNumber" xlink:label="loc_us-gaapShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingNumber_50" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="loc_us-gaapShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingNumber_50" xlink:type="arc" order="0" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingWeightedAverageExercisePrice" xlink:label="loc_us-gaapShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingWeightedAverageExercisePrice_50" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="loc_us-gaapShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingWeightedAverageExercisePrice_50" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardOptionsOutstandingWeightedAverageRemainingContractualTerm2" xlink:label="loc_us-gaapSharebasedCompensationArrangementBySharebasedPaymentAwardOptionsOutstandingWeightedAverageRemainingContractualTerm2_50" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="loc_us-gaapSharebasedCompensationArrangementBySharebasedPaymentAwardOptionsOutstandingWeightedAverageRemainingContractualTerm2_50" xlink:type="arc" order="2" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsGrantsInPeriodGross" xlink:label="loc_us-gaapShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsGrantsInPeriodGross_50" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="loc_us-gaapShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsGrantsInPeriodGross_50" xlink:type="arc" order="3" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardOptionsGrantsInPeriodWeightedAverageExercisePrice" xlink:label="loc_us-gaapShareBasedCompensationArrangementsByShareBasedPaymentAwardOptionsGrantsInPeriodWeightedAverageExercisePrice_50" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="loc_us-gaapShareBasedCompensationArrangementsByShareBasedPaymentAwardOptionsGrantsInPeriodWeightedAverageExercisePrice_50" xlink:type="arc" order="4" />
      <link:loc xlink:type="locator" xlink:href="ptix-20260630.xsd#PTIX_SharebasedCompensationArrangementBySharebasedPaymentAwardOptionsOutstandingWeightedAverageRemainingContractualGranted" xlink:label="loc_PTIXSharebasedCompensationArrangementBySharebasedPaymentAwardOptionsOutstandingWeightedAverageRemainingContractualGranted_50" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="loc_PTIXSharebasedCompensationArrangementBySharebasedPaymentAwardOptionsOutstandingWeightedAverageRemainingContractualGranted_50" xlink:type="arc" order="5" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsExpirationsInPeriod" xlink:label="loc_us-gaapShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsExpirationsInPeriod_50" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="loc_us-gaapShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsExpirationsInPeriod_50" xlink:type="arc" order="6" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardOptionsExpirationsInPeriodWeightedAverageExercisePrice" xlink:label="loc_us-gaapShareBasedCompensationArrangementsByShareBasedPaymentAwardOptionsExpirationsInPeriodWeightedAverageExercisePrice_50" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="loc_us-gaapShareBasedCompensationArrangementsByShareBasedPaymentAwardOptionsExpirationsInPeriodWeightedAverageExercisePrice_50" xlink:type="arc" order="7" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StockIssuedDuringPeriodSharesStockOptionsExercised" xlink:label="loc_us-gaapStockIssuedDuringPeriodSharesStockOptionsExercised_50" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="loc_us-gaapStockIssuedDuringPeriodSharesStockOptionsExercised_50" xlink:type="arc" order="8" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardOptionsExercisesInPeriodWeightedAverageExercisePrice" xlink:label="loc_us-gaapShareBasedCompensationArrangementsByShareBasedPaymentAwardOptionsExercisesInPeriodWeightedAverageExercisePrice_50" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="loc_us-gaapShareBasedCompensationArrangementsByShareBasedPaymentAwardOptionsExercisesInPeriodWeightedAverageExercisePrice_50" xlink:type="arc" order="9" />
    </link:definitionLink>
    <link:definitionLink xlink:type="extended" xlink:role="http://protagenic.com/role/ScheduleOfShare-basedCompensationNonvestedSharesDetails" xlink:title="999045 - Disclosure - SCHEDULE OF SHARE-BASED COMPENSATION NONVESTED SHARES (Details)">
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:label="loc_us-gaapShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable" xlink:label="loc_us-gaapScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xbrldt:contextElement="segment" xlink:from="loc_us-gaapShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="loc_us-gaapScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable" xlink:type="arc" order="10" xbrldt:closed="true" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AwardTypeAxis" xlink:label="loc_us-gaapAwardTypeAxis" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaapScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable" xlink:to="loc_us-gaapAwardTypeAxis" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:label="loc_us-gaapShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaapAwardTypeAxis" xlink:to="loc_us-gaapShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:type="arc" order="20" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:label="loc_us-gaapShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_10" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xbrldt:contextElement="segment" xlink:from="loc_us-gaapAwardTypeAxis" xlink:to="loc_us-gaapShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_10" xlink:type="arc" order="30" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_EmployeeStockOptionMember" xlink:label="loc_us-gaapEmployeeStockOptionMember_0" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:to="loc_us-gaapEmployeeStockOptionMember_0" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardOptionsNonvestedNumberOfShares" xlink:label="loc_us-gaapSharebasedCompensationArrangementBySharebasedPaymentAwardOptionsNonvestedNumberOfShares_40" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="loc_us-gaapSharebasedCompensationArrangementBySharebasedPaymentAwardOptionsNonvestedNumberOfShares_40" xlink:type="arc" order="0" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardOptionsNonvestedWeightedAverageGrantDateFairValue" xlink:label="loc_us-gaapSharebasedCompensationArrangementBySharebasedPaymentAwardOptionsNonvestedWeightedAverageGrantDateFairValue_40" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="loc_us-gaapSharebasedCompensationArrangementBySharebasedPaymentAwardOptionsNonvestedWeightedAverageGrantDateFairValue_40" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsGrantsInPeriodGross" xlink:label="loc_us-gaapShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsGrantsInPeriodGross_40" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="loc_us-gaapShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsGrantsInPeriodGross_40" xlink:type="arc" order="2" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsGrantsInPeriodWeightedAverageGrantDateFairValue" xlink:label="loc_us-gaapShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsGrantsInPeriodWeightedAverageGrantDateFairValue_40" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="loc_us-gaapShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsGrantsInPeriodWeightedAverageGrantDateFairValue_40" xlink:type="arc" order="3" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardOptionsVestedNumberOfShares" xlink:label="loc_us-gaapSharebasedCompensationArrangementBySharebasedPaymentAwardOptionsVestedNumberOfShares_40" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="loc_us-gaapSharebasedCompensationArrangementBySharebasedPaymentAwardOptionsVestedNumberOfShares_40" xlink:type="arc" order="4" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardOptionsVestedWeightedAverageGrantDateFairValue" xlink:label="loc_us-gaapSharebasedCompensationArrangementBySharebasedPaymentAwardOptionsVestedWeightedAverageGrantDateFairValue_40" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="loc_us-gaapSharebasedCompensationArrangementBySharebasedPaymentAwardOptionsVestedWeightedAverageGrantDateFairValue_40" xlink:type="arc" order="5" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsForfeituresInPeriod" xlink:label="loc_us-gaapShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsForfeituresInPeriod_40" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="loc_us-gaapShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsForfeituresInPeriod_40" xlink:type="arc" order="6" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardOptionsForfeituresInPeriodWeightedAverageExercisePrice" xlink:label="loc_us-gaapShareBasedCompensationArrangementsByShareBasedPaymentAwardOptionsForfeituresInPeriodWeightedAverageExercisePrice_40" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="loc_us-gaapShareBasedCompensationArrangementsByShareBasedPaymentAwardOptionsForfeituresInPeriodWeightedAverageExercisePrice_40" xlink:type="arc" order="7" />
    </link:definitionLink>
    <link:definitionLink xlink:type="extended" xlink:role="http://protagenic.com/role/SummaryOfWarrantDetails" xlink:title="999046 - Disclosure - SUMMARY OF WARRANT (Details)">
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AccumulatedOtherComprehensiveIncomeLossLineItems" xlink:label="loc_us-gaapAccumulatedOtherComprehensiveIncomeLossLineItems" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AccumulatedOtherComprehensiveIncomeLossTable" xlink:label="loc_us-gaapAccumulatedOtherComprehensiveIncomeLossTable" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xbrldt:contextElement="segment" xlink:from="loc_us-gaapAccumulatedOtherComprehensiveIncomeLossLineItems" xlink:to="loc_us-gaapAccumulatedOtherComprehensiveIncomeLossTable" xlink:type="arc" order="10" xbrldt:closed="true" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementEquityComponentsAxis" xlink:label="loc_us-gaapStatementEquityComponentsAxis" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaapAccumulatedOtherComprehensiveIncomeLossTable" xlink:to="loc_us-gaapStatementEquityComponentsAxis" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_EquityComponentDomain" xlink:label="loc_us-gaapEquityComponentDomain" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaapStatementEquityComponentsAxis" xlink:to="loc_us-gaapEquityComponentDomain" xlink:type="arc" order="20" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_EquityComponentDomain" xlink:label="loc_us-gaapEquityComponentDomain_10" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xbrldt:contextElement="segment" xlink:from="loc_us-gaapStatementEquityComponentsAxis" xlink:to="loc_us-gaapEquityComponentDomain_10" xlink:type="arc" order="30" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_WarrantMember" xlink:label="loc_us-gaapWarrantMember_0" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapEquityComponentDomain" xlink:to="loc_us-gaapWarrantMember_0" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardNonOptionEquityInstrumentsOutstandingNumber" xlink:label="loc_us-gaapShareBasedCompensationArrangementByShareBasedPaymentAwardNonOptionEquityInstrumentsOutstandingNumber_50" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapAccumulatedOtherComprehensiveIncomeLossLineItems" xlink:to="loc_us-gaapShareBasedCompensationArrangementByShareBasedPaymentAwardNonOptionEquityInstrumentsOutstandingNumber_50" xlink:type="arc" order="0" />
      <link:loc xlink:type="locator" xlink:href="ptix-20260630.xsd#PTIX_ShareBasedCompensationArrangementByShareBasedPaymentAwardNonOptionEquityInstrumentsOutstandingWeightedAverageExercisePrice" xlink:label="loc_PTIXShareBasedCompensationArrangementByShareBasedPaymentAwardNonOptionEquityInstrumentsOutstandingWeightedAverageExercisePrice_50" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapAccumulatedOtherComprehensiveIncomeLossLineItems" xlink:to="loc_PTIXShareBasedCompensationArrangementByShareBasedPaymentAwardNonOptionEquityInstrumentsOutstandingWeightedAverageExercisePrice_50" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="ptix-20260630.xsd#PTIX_ShareBasedCompensationArrangementByShareBasedPaymentAwardNonOptionEquityInstrumentsOutstandingWeightedAverageRemainingContractualTerm" xlink:label="loc_PTIXShareBasedCompensationArrangementByShareBasedPaymentAwardNonOptionEquityInstrumentsOutstandingWeightedAverageRemainingContractualTerm_50" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapAccumulatedOtherComprehensiveIncomeLossLineItems" xlink:to="loc_PTIXShareBasedCompensationArrangementByShareBasedPaymentAwardNonOptionEquityInstrumentsOutstandingWeightedAverageRemainingContractualTerm_50" xlink:type="arc" order="2" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardNonOptionEquityInstrumentsGranted" xlink:label="loc_us-gaapShareBasedCompensationArrangementByShareBasedPaymentAwardNonOptionEquityInstrumentsGranted_50" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapAccumulatedOtherComprehensiveIncomeLossLineItems" xlink:to="loc_us-gaapShareBasedCompensationArrangementByShareBasedPaymentAwardNonOptionEquityInstrumentsGranted_50" xlink:type="arc" order="3" />
      <link:loc xlink:type="locator" xlink:href="ptix-20260630.xsd#PTIX_ShareBasedCompensationArrangementByShareBasedPaymentAwardNonOptionEquityInstrumentsGrantsInPeriodWeightedAverageExercisePrice" xlink:label="loc_PTIXShareBasedCompensationArrangementByShareBasedPaymentAwardNonOptionEquityInstrumentsGrantsInPeriodWeightedAverageExercisePrice_50" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapAccumulatedOtherComprehensiveIncomeLossLineItems" xlink:to="loc_PTIXShareBasedCompensationArrangementByShareBasedPaymentAwardNonOptionEquityInstrumentsGrantsInPeriodWeightedAverageExercisePrice_50" xlink:type="arc" order="4" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardNonOptionEquityInstrumentsExpirations" xlink:label="loc_us-gaapShareBasedCompensationArrangementByShareBasedPaymentAwardNonOptionEquityInstrumentsExpirations_50" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapAccumulatedOtherComprehensiveIncomeLossLineItems" xlink:to="loc_us-gaapShareBasedCompensationArrangementByShareBasedPaymentAwardNonOptionEquityInstrumentsExpirations_50" xlink:type="arc" order="5" />
      <link:loc xlink:type="locator" xlink:href="ptix-20260630.xsd#PTIX_ShareBasedCompensationArrangementByShareBasedPaymentAwardNonOptionEquityInstrumentsExpirationsInPeriodWeightedAverageExercisePrice" xlink:label="loc_PTIXShareBasedCompensationArrangementByShareBasedPaymentAwardNonOptionEquityInstrumentsExpirationsInPeriodWeightedAverageExercisePrice_50" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapAccumulatedOtherComprehensiveIncomeLossLineItems" xlink:to="loc_PTIXShareBasedCompensationArrangementByShareBasedPaymentAwardNonOptionEquityInstrumentsExpirationsInPeriodWeightedAverageExercisePrice_50" xlink:type="arc" order="6" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardNonOptionEquityInstrumentsExercised" xlink:label="loc_us-gaapShareBasedCompensationArrangementByShareBasedPaymentAwardNonOptionEquityInstrumentsExercised_50" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapAccumulatedOtherComprehensiveIncomeLossLineItems" xlink:to="loc_us-gaapShareBasedCompensationArrangementByShareBasedPaymentAwardNonOptionEquityInstrumentsExercised_50" xlink:type="arc" order="7" />
      <link:loc xlink:type="locator" xlink:href="ptix-20260630.xsd#PTIX_ShareBasedCompensationArrangementByShareBasedPaymentAwardNonOptionEquityInstrumentsExercisedInPeriodWeightedAverageExercisePrice" xlink:label="loc_PTIXShareBasedCompensationArrangementByShareBasedPaymentAwardNonOptionEquityInstrumentsExercisedInPeriodWeightedAverageExercisePrice_50" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapAccumulatedOtherComprehensiveIncomeLossLineItems" xlink:to="loc_PTIXShareBasedCompensationArrangementByShareBasedPaymentAwardNonOptionEquityInstrumentsExercisedInPeriodWeightedAverageExercisePrice_50" xlink:type="arc" order="8" />
    </link:definitionLink>
    <link:definitionLink xlink:type="extended" xlink:role="http://protagenic.com/role/StockholdersEquityDetailsNarrative" xlink:title="999047 - Disclosure - STOCKHOLDERS&#8217; EQUITY (Details Narrative)">
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AccumulatedOtherComprehensiveIncomeLossLineItems" xlink:label="loc_us-gaapAccumulatedOtherComprehensiveIncomeLossLineItems" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AccumulatedOtherComprehensiveIncomeLossTable" xlink:label="loc_us-gaapAccumulatedOtherComprehensiveIncomeLossTable" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xbrldt:contextElement="segment" xlink:from="loc_us-gaapAccumulatedOtherComprehensiveIncomeLossLineItems" xlink:to="loc_us-gaapAccumulatedOtherComprehensiveIncomeLossTable" xlink:type="arc" order="10" xbrldt:closed="true" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementEquityComponentsAxis" xlink:label="loc_us-gaapStatementEquityComponentsAxis" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaapAccumulatedOtherComprehensiveIncomeLossTable" xlink:to="loc_us-gaapStatementEquityComponentsAxis" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_EquityComponentDomain" xlink:label="loc_us-gaapEquityComponentDomain" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaapStatementEquityComponentsAxis" xlink:to="loc_us-gaapEquityComponentDomain" xlink:type="arc" order="20" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_EquityComponentDomain" xlink:label="loc_us-gaapEquityComponentDomain_10" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xbrldt:contextElement="segment" xlink:from="loc_us-gaapStatementEquityComponentsAxis" xlink:to="loc_us-gaapEquityComponentDomain_10" xlink:type="arc" order="30" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_CommonStockMember" xlink:label="loc_us-gaapCommonStockMember_0" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapEquityComponentDomain" xlink:to="loc_us-gaapCommonStockMember_0" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_WarrantMember" xlink:label="loc_us-gaapWarrantMember_50" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapEquityComponentDomain" xlink:to="loc_us-gaapWarrantMember_50" xlink:type="arc" order="51" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_BusinessAcquisitionAxis" xlink:label="loc_us-gaapBusinessAcquisitionAxis" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaapAccumulatedOtherComprehensiveIncomeLossTable" xlink:to="loc_us-gaapBusinessAcquisitionAxis" xlink:type="arc" order="2" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_BusinessAcquisitionAcquireeDomain" xlink:label="loc_us-gaapBusinessAcquisitionAcquireeDomain" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaapBusinessAcquisitionAxis" xlink:to="loc_us-gaapBusinessAcquisitionAcquireeDomain" xlink:type="arc" order="20" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_BusinessAcquisitionAcquireeDomain" xlink:label="loc_us-gaapBusinessAcquisitionAcquireeDomain_10" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xbrldt:contextElement="segment" xlink:from="loc_us-gaapBusinessAcquisitionAxis" xlink:to="loc_us-gaapBusinessAcquisitionAcquireeDomain_10" xlink:type="arc" order="30" />
      <link:loc xlink:type="locator" xlink:href="ptix-20260630.xsd#PTIX_PhytanixBioMember" xlink:label="loc_PTIXPhytanixBioMember_0" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapBusinessAcquisitionAcquireeDomain" xlink:to="loc_PTIXPhytanixBioMember_0" xlink:type="arc" order="2" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementClassOfStockAxis" xlink:label="loc_us-gaapStatementClassOfStockAxis" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaapAccumulatedOtherComprehensiveIncomeLossTable" xlink:to="loc_us-gaapStatementClassOfStockAxis" xlink:type="arc" order="3" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfStockDomain" xlink:label="loc_us-gaapClassOfStockDomain" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaapStatementClassOfStockAxis" xlink:to="loc_us-gaapClassOfStockDomain" xlink:type="arc" order="20" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfStockDomain" xlink:label="loc_us-gaapClassOfStockDomain_10" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xbrldt:contextElement="segment" xlink:from="loc_us-gaapStatementClassOfStockAxis" xlink:to="loc_us-gaapClassOfStockDomain_10" xlink:type="arc" order="30" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SeriesCPreferredStockMember" xlink:label="loc_us-gaapSeriesCPreferredStockMember_10" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapClassOfStockDomain" xlink:to="loc_us-gaapSeriesCPreferredStockMember_10" xlink:type="arc" order="11" />
      <link:loc xlink:type="locator" xlink:href="ptix-20260630.xsd#PTIX_SeriesCOnePreferredStockMember" xlink:label="loc_PTIXSeriesCOnePreferredStockMember_20" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapClassOfStockDomain" xlink:to="loc_PTIXSeriesCOnePreferredStockMember_20" xlink:type="arc" order="21" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SeriesDPreferredStockMember" xlink:label="loc_us-gaapSeriesDPreferredStockMember_30" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapClassOfStockDomain" xlink:to="loc_us-gaapSeriesDPreferredStockMember_30" xlink:type="arc" order="31" />
      <link:loc xlink:type="locator" xlink:href="ptix-20260630.xsd#PTIX_SeriesC1ConvertiblePreferredStockMember" xlink:label="loc_PTIXSeriesC1ConvertiblePreferredStockMember_80" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapClassOfStockDomain" xlink:to="loc_PTIXSeriesC1ConvertiblePreferredStockMember_80" xlink:type="arc" order="81" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AwardTypeAxis" xlink:label="loc_us-gaapAwardTypeAxis" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaapAccumulatedOtherComprehensiveIncomeLossTable" xlink:to="loc_us-gaapAwardTypeAxis" xlink:type="arc" order="4" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:label="loc_us-gaapShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaapAwardTypeAxis" xlink:to="loc_us-gaapShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:type="arc" order="20" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:label="loc_us-gaapShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_10" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xbrldt:contextElement="segment" xlink:from="loc_us-gaapAwardTypeAxis" xlink:to="loc_us-gaapShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_10" xlink:type="arc" order="30" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_EmployeeStockOptionMember" xlink:label="loc_us-gaapEmployeeStockOptionMember_100" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:to="loc_us-gaapEmployeeStockOptionMember_100" xlink:type="arc" order="101" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_TitleOfIndividualAxis" xlink:label="loc_srtTitleOfIndividualAxis" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaapAccumulatedOtherComprehensiveIncomeLossTable" xlink:to="loc_srtTitleOfIndividualAxis" xlink:type="arc" order="5" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_TitleOfIndividualWithRelationshipToEntityDomain" xlink:label="loc_srtTitleOfIndividualWithRelationshipToEntityDomain" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_srtTitleOfIndividualAxis" xlink:to="loc_srtTitleOfIndividualWithRelationshipToEntityDomain" xlink:type="arc" order="20" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_TitleOfIndividualWithRelationshipToEntityDomain" xlink:label="loc_srtTitleOfIndividualWithRelationshipToEntityDomain_10" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xbrldt:contextElement="segment" xlink:from="loc_srtTitleOfIndividualAxis" xlink:to="loc_srtTitleOfIndividualWithRelationshipToEntityDomain_10" xlink:type="arc" order="30" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_PresidentMember" xlink:label="loc_srtPresidentMember_140" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srtTitleOfIndividualWithRelationshipToEntityDomain" xlink:to="loc_srtPresidentMember_140" xlink:type="arc" order="141" />
      <link:loc xlink:type="locator" xlink:href="ptix-20260630.xsd#PTIX_EmployeesMember" xlink:label="loc_PTIXEmployeesMember_150" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srtTitleOfIndividualWithRelationshipToEntityDomain" xlink:to="loc_PTIXEmployeesMember_150" xlink:type="arc" order="151" />
      <link:loc xlink:type="locator" xlink:href="ptix-20260630.xsd#PTIX_NonEmployeesMember" xlink:label="loc_PTIXNonEmployeesMember_170" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srtTitleOfIndividualWithRelationshipToEntityDomain" xlink:to="loc_PTIXNonEmployeesMember_170" xlink:type="arc" order="171" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StockIssuedDuringPeriodSharesAcquisitions" xlink:label="loc_us-gaapStockIssuedDuringPeriodSharesAcquisitions_250" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapAccumulatedOtherComprehensiveIncomeLossLineItems" xlink:to="loc_us-gaapStockIssuedDuringPeriodSharesAcquisitions_250" xlink:type="arc" order="0" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StockIssuedDuringPeriodSharesNewIssues" xlink:label="loc_us-gaapStockIssuedDuringPeriodSharesNewIssues_250" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapAccumulatedOtherComprehensiveIncomeLossLineItems" xlink:to="loc_us-gaapStockIssuedDuringPeriodSharesNewIssues_250" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StockIssuedDuringPeriodValueNewIssues" xlink:label="loc_us-gaapStockIssuedDuringPeriodValueNewIssues_250" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapAccumulatedOtherComprehensiveIncomeLossLineItems" xlink:to="loc_us-gaapStockIssuedDuringPeriodValueNewIssues_250" xlink:type="arc" order="2" />
      <link:loc xlink:type="locator" xlink:href="ptix-20260630.xsd#PTIX_StockIssuedDuringPeriodSharesWarrantExercise" xlink:label="loc_PTIXStockIssuedDuringPeriodSharesWarrantExercise_250" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapAccumulatedOtherComprehensiveIncomeLossLineItems" xlink:to="loc_PTIXStockIssuedDuringPeriodSharesWarrantExercise_250" xlink:type="arc" order="3" />
      <link:loc xlink:type="locator" xlink:href="ptix-20260630.xsd#PTIX_StockIssuedDuringPeriodValueWarrantExercise" xlink:label="loc_PTIXStockIssuedDuringPeriodValueWarrantExercise_250" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapAccumulatedOtherComprehensiveIncomeLossLineItems" xlink:to="loc_PTIXStockIssuedDuringPeriodValueWarrantExercise_250" xlink:type="arc" order="4" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StockIssuedDuringPeriodSharesReverseStockSplits" xlink:label="loc_us-gaapStockIssuedDuringPeriodSharesReverseStockSplits_250" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapAccumulatedOtherComprehensiveIncomeLossLineItems" xlink:to="loc_us-gaapStockIssuedDuringPeriodSharesReverseStockSplits_250" xlink:type="arc" order="5" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_PreferredStockConvertibleSharesIssuable" xlink:label="loc_us-gaapPreferredStockConvertibleSharesIssuable_250" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapAccumulatedOtherComprehensiveIncomeLossLineItems" xlink:to="loc_us-gaapPreferredStockConvertibleSharesIssuable_250" xlink:type="arc" order="6" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_PreferredStockConvertibleConversionPrice" xlink:label="loc_us-gaapPreferredStockConvertibleConversionPrice_250" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapAccumulatedOtherComprehensiveIncomeLossLineItems" xlink:to="loc_us-gaapPreferredStockConvertibleConversionPrice_250" xlink:type="arc" order="7" />
      <link:loc xlink:type="locator" xlink:href="ptix-20260630.xsd#PTIX_PreferredStockStatedValue" xlink:label="loc_PTIXPreferredStockStatedValue_250" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapAccumulatedOtherComprehensiveIncomeLossLineItems" xlink:to="loc_PTIXPreferredStockStatedValue_250" xlink:type="arc" order="8" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingNumber" xlink:label="loc_us-gaapShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingNumber_250" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapAccumulatedOtherComprehensiveIncomeLossLineItems" xlink:to="loc_us-gaapShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingNumber_250" xlink:type="arc" order="9" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsGrantsInPeriodGross" xlink:label="loc_us-gaapShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsGrantsInPeriodGross_250" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapAccumulatedOtherComprehensiveIncomeLossLineItems" xlink:to="loc_us-gaapShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsGrantsInPeriodGross_250" xlink:type="arc" order="10" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingWeightedAverageExercisePrice" xlink:label="loc_us-gaapShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingWeightedAverageExercisePrice_250" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapAccumulatedOtherComprehensiveIncomeLossLineItems" xlink:to="loc_us-gaapShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingWeightedAverageExercisePrice_250" xlink:type="arc" order="11" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingIntrinsicValue" xlink:label="loc_us-gaapShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingIntrinsicValue_250" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapAccumulatedOtherComprehensiveIncomeLossLineItems" xlink:to="loc_us-gaapShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingIntrinsicValue_250" xlink:type="arc" order="12" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsGrantsInPeriodWeightedAverageGrantDateFairValue" xlink:label="loc_us-gaapShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsGrantsInPeriodWeightedAverageGrantDateFairValue_250" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapAccumulatedOtherComprehensiveIncomeLossLineItems" xlink:to="loc_us-gaapShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsGrantsInPeriodWeightedAverageGrantDateFairValue_250" xlink:type="arc" order="13" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardAwardVestingPeriod1" xlink:label="loc_us-gaapShareBasedCompensationArrangementByShareBasedPaymentAwardAwardVestingPeriod1_250" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapAccumulatedOtherComprehensiveIncomeLossLineItems" xlink:to="loc_us-gaapShareBasedCompensationArrangementByShareBasedPaymentAwardAwardVestingPeriod1_250" xlink:type="arc" order="14" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardExpirationPeriod" xlink:label="loc_us-gaapSharebasedCompensationArrangementBySharebasedPaymentAwardExpirationPeriod_250" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapAccumulatedOtherComprehensiveIncomeLossLineItems" xlink:to="loc_us-gaapSharebasedCompensationArrangementBySharebasedPaymentAwardExpirationPeriod_250" xlink:type="arc" order="15" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AllocatedShareBasedCompensationExpense" xlink:label="loc_us-gaapAllocatedShareBasedCompensationExpense_250" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapAccumulatedOtherComprehensiveIncomeLossLineItems" xlink:to="loc_us-gaapAllocatedShareBasedCompensationExpense_250" xlink:type="arc" order="16" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_EmployeeServiceShareBasedCompensationNonvestedAwardsTotalCompensationCostNotYetRecognizedStockOptions" xlink:label="loc_us-gaapEmployeeServiceShareBasedCompensationNonvestedAwardsTotalCompensationCostNotYetRecognizedStockOptions_250" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapAccumulatedOtherComprehensiveIncomeLossLineItems" xlink:to="loc_us-gaapEmployeeServiceShareBasedCompensationNonvestedAwardsTotalCompensationCostNotYetRecognizedStockOptions_250" xlink:type="arc" order="17" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_EmployeeServiceShareBasedCompensationNonvestedAwardsTotalCompensationCostNotYetRecognizedPeriodForRecognition1" xlink:label="loc_us-gaapEmployeeServiceShareBasedCompensationNonvestedAwardsTotalCompensationCostNotYetRecognizedPeriodForRecognition1_250" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapAccumulatedOtherComprehensiveIncomeLossLineItems" xlink:to="loc_us-gaapEmployeeServiceShareBasedCompensationNonvestedAwardsTotalCompensationCostNotYetRecognizedPeriodForRecognition1_250" xlink:type="arc" order="18" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardNonOptionEquityInstrumentsOutstandingNumber" xlink:label="loc_us-gaapShareBasedCompensationArrangementByShareBasedPaymentAwardNonOptionEquityInstrumentsOutstandingNumber_250" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapAccumulatedOtherComprehensiveIncomeLossLineItems" xlink:to="loc_us-gaapShareBasedCompensationArrangementByShareBasedPaymentAwardNonOptionEquityInstrumentsOutstandingNumber_250" xlink:type="arc" order="19" />
      <link:loc xlink:type="locator" xlink:href="ptix-20260630.xsd#PTIX_ShareBasedCompensationArrangementByShareBasedPaymentAwardNonOptionEquityInstrumentsOutstandingWeightedAverageExercisePrice" xlink:label="loc_PTIXShareBasedCompensationArrangementByShareBasedPaymentAwardNonOptionEquityInstrumentsOutstandingWeightedAverageExercisePrice_250" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapAccumulatedOtherComprehensiveIncomeLossLineItems" xlink:to="loc_PTIXShareBasedCompensationArrangementByShareBasedPaymentAwardNonOptionEquityInstrumentsOutstandingWeightedAverageExercisePrice_250" xlink:type="arc" order="20" />
      <link:loc xlink:type="locator" xlink:href="ptix-20260630.xsd#PTIX_SharebasedCompensationArrangementBySharebasedPaymentAwardEquityInstrumentsNonOptionsAggregateIntrinsicValueOutstanding" xlink:label="loc_PTIXSharebasedCompensationArrangementBySharebasedPaymentAwardEquityInstrumentsNonOptionsAggregateIntrinsicValueOutstanding_250" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapAccumulatedOtherComprehensiveIncomeLossLineItems" xlink:to="loc_PTIXSharebasedCompensationArrangementBySharebasedPaymentAwardEquityInstrumentsNonOptionsAggregateIntrinsicValueOutstanding_250" xlink:type="arc" order="21" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_EmployeeBenefitsAndShareBasedCompensation" xlink:label="loc_us-gaapEmployeeBenefitsAndShareBasedCompensation_250" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapAccumulatedOtherComprehensiveIncomeLossLineItems" xlink:to="loc_us-gaapEmployeeBenefitsAndShareBasedCompensation_250" xlink:type="arc" order="22" />
    </link:definitionLink>
    <link:definitionLink xlink:type="extended" xlink:role="http://protagenic.com/role/CollaborativeAgreementsDetailsNarrative" xlink:title="999048 - Disclosure - COLLABORATIVE AGREEMENTS (Details Narrative)">
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:label="loc_us-gaapCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ScheduleOfCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsTable" xlink:label="loc_us-gaapScheduleOfCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsTable" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xbrldt:contextElement="segment" xlink:from="loc_us-gaapCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:to="loc_us-gaapScheduleOfCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsTable" xlink:type="arc" order="10" xbrldt:closed="true" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_TypeOfArrangementAxis" xlink:label="loc_us-gaapTypeOfArrangementAxis" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaapScheduleOfCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsTable" xlink:to="loc_us-gaapTypeOfArrangementAxis" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:label="loc_us-gaapArrangementsAndNonarrangementTransactionsMember" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaapTypeOfArrangementAxis" xlink:to="loc_us-gaapArrangementsAndNonarrangementTransactionsMember" xlink:type="arc" order="20" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:label="loc_us-gaapArrangementsAndNonarrangementTransactionsMember_10" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xbrldt:contextElement="segment" xlink:from="loc_us-gaapTypeOfArrangementAxis" xlink:to="loc_us-gaapArrangementsAndNonarrangementTransactionsMember_10" xlink:type="arc" order="30" />
      <link:loc xlink:type="locator" xlink:href="ptix-20260630.xsd#PTIX_ResearchAgreementsMember" xlink:label="loc_PTIXResearchAgreementsMember_0" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapArrangementsAndNonarrangementTransactionsMember" xlink:to="loc_PTIXResearchAgreementsMember_0" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ResearchAndDevelopmentExpense" xlink:label="loc_us-gaapResearchAndDevelopmentExpense_30" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:to="loc_us-gaapResearchAndDevelopmentExpense_30" xlink:type="arc" order="0" />
    </link:definitionLink>
    <link:definitionLink xlink:type="extended" xlink:role="http://protagenic.com/role/CommitmentsAndContingenciesDetailsNarrative" xlink:title="999049 - Disclosure - COMMITMENTS AND CONTINGENCIES (Details Narrative)">
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_FiniteLivedIntangibleAssetsLineItems" xlink:label="loc_us-gaapFiniteLivedIntangibleAssetsLineItems" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ScheduleOfFiniteLivedIntangibleAssetsTable" xlink:label="loc_us-gaapScheduleOfFiniteLivedIntangibleAssetsTable" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xbrldt:contextElement="segment" xlink:from="loc_us-gaapFiniteLivedIntangibleAssetsLineItems" xlink:to="loc_us-gaapScheduleOfFiniteLivedIntangibleAssetsTable" xlink:type="arc" order="10" xbrldt:closed="true" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_FiniteLivedIntangibleAssetsByMajorClassAxis" xlink:label="loc_us-gaapFiniteLivedIntangibleAssetsByMajorClassAxis" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaapScheduleOfFiniteLivedIntangibleAssetsTable" xlink:to="loc_us-gaapFiniteLivedIntangibleAssetsByMajorClassAxis" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_FiniteLivedIntangibleAssetsMajorClassNameDomain" xlink:label="loc_us-gaapFiniteLivedIntangibleAssetsMajorClassNameDomain" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaapFiniteLivedIntangibleAssetsByMajorClassAxis" xlink:to="loc_us-gaapFiniteLivedIntangibleAssetsMajorClassNameDomain" xlink:type="arc" order="20" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_FiniteLivedIntangibleAssetsMajorClassNameDomain" xlink:label="loc_us-gaapFiniteLivedIntangibleAssetsMajorClassNameDomain_10" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xbrldt:contextElement="segment" xlink:from="loc_us-gaapFiniteLivedIntangibleAssetsByMajorClassAxis" xlink:to="loc_us-gaapFiniteLivedIntangibleAssetsMajorClassNameDomain_10" xlink:type="arc" order="30" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_LicensingAgreementsMember" xlink:label="loc_us-gaapLicensingAgreementsMember_0" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapFiniteLivedIntangibleAssetsMajorClassNameDomain" xlink:to="loc_us-gaapLicensingAgreementsMember_0" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="ptix-20260630.xsd#PTIX_RoyaltyPaymentPercentage" xlink:label="loc_PTIXRoyaltyPaymentPercentage_70" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapFiniteLivedIntangibleAssetsLineItems" xlink:to="loc_PTIXRoyaltyPaymentPercentage_70" xlink:type="arc" order="0" />
      <link:loc xlink:type="locator" xlink:href="ptix-20260630.xsd#PTIX_UpFrontSubLicenseFeesPercentage" xlink:label="loc_PTIXUpFrontSubLicenseFeesPercentage_70" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapFiniteLivedIntangibleAssetsLineItems" xlink:to="loc_PTIXUpFrontSubLicenseFeesPercentage_70" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="ptix-20260630.xsd#PTIX_RoyaltyPaymentOnBehalfOfSublicenseePercentage" xlink:label="loc_PTIXRoyaltyPaymentOnBehalfOfSublicenseePercentage_70" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapFiniteLivedIntangibleAssetsLineItems" xlink:to="loc_PTIXRoyaltyPaymentOnBehalfOfSublicenseePercentage_70" xlink:type="arc" order="2" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_Revenues" xlink:label="loc_us-gaapRevenues_70" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapFiniteLivedIntangibleAssetsLineItems" xlink:to="loc_us-gaapRevenues_70" xlink:type="arc" order="3" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SharePrice" xlink:label="loc_us-gaapSharePrice_70" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapFiniteLivedIntangibleAssetsLineItems" xlink:to="loc_us-gaapSharePrice_70" xlink:type="arc" order="4" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StockholdersEquityReverseStockSplit" xlink:label="loc_us-gaapStockholdersEquityReverseStockSplit_70" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapFiniteLivedIntangibleAssetsLineItems" xlink:to="loc_us-gaapStockholdersEquityReverseStockSplit_70" xlink:type="arc" order="5" />
    </link:definitionLink>
    <link:definitionLink xlink:type="extended" xlink:role="http://protagenic.com/role/SegmentReportingDetailsNarrative" xlink:title="999050 - Disclosure - SEGMENT REPORTING (Details Narrative)" />
</link:linkbase>
