Assessment instruments designed to measure student conceptual understanding and skills proficiency related to biochemistry are important to transform undergraduate biochemistry education. The purpose of this study was to develop an assessment instrument to measure student understanding of protein structure and enzyme inhibition in a new context, that of saturable binding. A community of biochemistry educators was involved in an iterative process of designing and testing of this assessment, which consists of true/false and open-ended questions that map to low and high levels in Bloom's taxonomy. A total of 188 students' responses were collected from seven different institutions and were graded by two independent raters using a rubric. Results from this administration indicate that most students were able to answer the questions related to lower-levels in Bloom's taxonomy; however for higher-level questions, students had more difficulty. The results from this assessment can give instructors insight into the ways in which persistent incorrect or incomplete ideas related to protein structure and binding events can hinder knowledge application. Also, the use of a community of practice to develop assessment instruments of this type and the format of the instrument itself could be a useful model for development of assessment instruments in the future.