The ability to separate, identify, and quantify proteins from complex mixtures are key foundational methods across biochemistry teaching and research. In particular, enzyme-linked immunosorbent assay (ELISA) is an important technique that is used to measure antigen concentrations in both industry and academia. There are four categories of ELISA, direct, indirect, competitive, and sandwich, each with their own applications. Sandwich ELISAs are used to determine antigen concentrations from complex mixtures of protein, such as a cell lysates, and are regularly used as medical diagnostics to diagnose illness and diseases ranging from hepatitis to celiac disease. One major problem with teaching the sandwich ELISA technique to students is the prohibitive cost due to the need to coat a 96-well plate with a capture antibody. One solution to this problem would be to significantly reduce the role of each student in the lab, but this does not adequately prepare students to perform the procedure in a research or industry lab. Instead, this laboratory exercise teaches students the procedural knowledge needed to perform a direct sandwich ELISA, but uses a simulated experience performed within a wet-lab environment. The presented scenario is the analysis of phosphorylated proteins within a synthetic signaling pathway, but because the lab uses simulated samples, it can be tailored to different topics and educational aims. The procedure is 10- to 26-fold less expensive per student to deploy than an authentic sandwich ELISA. Students in the course report that the ELISA lab significantly strengthened the connection between theory and practice. © 2019 International Union of Biochemistry and Molecular Biology, 2019.