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Students in upper-division biochemistry laboratories are often not exposed to biochemical techniques that allow them to understand and measure binding equilibrium constants for protein interactions due to the specialized nature of the instruments required or the time constraints of the undergraduate laboratory. Here, we describe a 3-4 hour guided-inquiry laboratory module designed to measure the binding anities (KD) of the enzyme lactate dehydrogenase for two of its substrates, NADH and NAD+. These measurements are carried out using fluorescence techniques and a microplate reader to minimize reagent use and maximize the number of replicates. This laboratory is inexpensive, forgiving for students, and exhibits strong gains from students in their understanding of protein binding and experimental design for in vitro binding experiments.