Abstract Background and objectives Universities throughout the USA increasingly offer undergraduate courses in evolutionary medicine (EvMed), which creates a need for pedagogical resources. Several resources offer course content (e.g. textbooks) and a previous study identified EvMed core principles to help instructors set learning goals. However, assessment tools are not yet available. In this study, we address this need by developing an assessment that measures students’ ability to apply EvMed core principles to various health-related scenarios. Methodology The EvMed Assessment (EMA) consists of questions containing a short description of a health-related scenario followed by several likely/unlikely items. We evaluated the assessment’s validity and reliability using a variety of qualitative (expert reviews and student interviews) and quantitative (Cronbach’s α and classical test theory) methods. We iteratively revised the assessment through several rounds of validation. We then administered the assessment to undergraduates in EvMed and Evolution courses at multiple institutions. Results We used results from the pilot to create the EMA final draft. After conducting quantitative validation, we deleted items that failed to meet performance criteria and revised items that exhibited borderline performance. The final version of the EMA consists of six core questions containing 25 items, and five supplemental questions containing 20 items. Conclusions and implications The EMA is a pedagogical tool supported by a wide range of validation evidence. Instructors can use it as a pre/post measure of student learning in an EvMed course to inform curriculum revision, or as a test bank to draw upon when developing in-class assessments, quizzes or exams.