We describe the development and initial validity assessment of the 20-item BioCalculus Assessment (BCA), with the objective of comparing undergraduate life science students’ understanding of calculus concepts in different courses with alternative emphases (with and without focus on biological applications). The development process of the BCA included obtaining input from a large network of scientists and educators as well as students in calculus and biocalculus courses to accumulate evidential support of the instrument’s content validity and response processes of test takers. We used the Rasch model to examine the internal structure of scores from students who have experienced calculus instruction in the two methods. The analysis involved three populations (Calculus 1, Calculus 2, and Biocalculus) for which the Calc 1 and Calc 2 students were not exposed to calculus concepts in a life science setting, while the Biocalculus students were presented concepts explicitly with a life science emphasis. Overall, our findings indicate that the BCA has reasonable validity properties, providing a diagnostic tool to assess the relative learning success and calculus comprehension of undergraduate biology majors from alternative methods of instruction that do or do not emphasize life science examples.