<?xml version="1.0" encoding="US-ASCII"?>
<!-- Generated by Broadridge Transform (tm) - http://www.broadridge.com -->
<!-- Created: Wed Jun 03 21:23:10 UTC 2026 -->
<linkbase xmlns="http://www.xbrl.org/2003/linkbase" xmlns:xlink="http://www.w3.org/1999/xlink" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" xsi:schemaLocation="http://www.xbrl.org/2003/linkbase http://www.xbrl.org/2003/xbrl-linkbase-2003-12-31.xsd" xmlns:xbrldt="http://xbrl.org/2005/xbrldt">
  <roleRef xlink:type="simple" xlink:href="amst-20260603.xsd#amst_r_AdditionalDimensions" roleURI="http://amesite.com/role/AdditionalDimensions"/>
  <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"/>
  <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"/>
  <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"/>
  <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"/>
  <arcroleRef xlink:type="simple" xlink:href="http://www.xbrl.org/2005/xbrldt-2005.xsd#all" arcroleURI="http://xbrl.org/int/dim/arcrole/all"/>
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  <definitionLink xlink:role="http://amesite.com/role/AdditionalDimensions" xlink:type="extended"/>
</linkbase>
