Interest in student conception of experimentation inspired the development of a fully validated 14-question inventory on experimental design in biology (BEDCI) by following established best practices in concept inventory (CI) design. This CI can be used to diagnose specific examples of non-expert-like thinking in students and to evaluate the success of teaching strategies that target conceptual changes. We used BEDCI to diagnose non-expert-like student thinking in experimental design at the pre- and posttest stage in five courses (total n = 580 students) at a large research university in western Canada. Calculated difficulty and discrimination metrics indicated that BEDCI questions are able to effectively capture learning changes at the undergraduate level. A high correlation (r = 0.84) between responses by students in similar courses and at the same stage of their academic career, also suggests that the test is reliable. Students showed significant positive learning changes by the posttest stage, but some non-expert-like responses were widespread and persistent. BEDCI is a reliable and valid diagnostic tool that can be used in a variety of life sciences disciplines.