Enzyme function is central to student understanding of multiple topics within the biochemistry curriculum. In particular, students must understand how enzymes and substrates interact with one another. This manuscript describes the development of a 15-item Enzyme-Substrate Interactions Concept Inventory (ESICI) that measures student understanding of enzyme-substrate interactions. The validity and reliability of ESICI data were established through multiple methods. Results from the administration of the ESICI to biochemistry students across the United States (N = 707) are discussed in terms of instrument quality. The manuscript concludes with suggestions for how to use the ESICI for both teaching and biochemistry education research. (Contains 4 tables and 1 figure.)