Most biochemical transformations involve more than one substrate. Bisubstrate enzymes catalyze multiple chemical reactions in living systems and include members of the transferase, oxidoreductase, and ligase enzyme classes. Working knowledge of bisubstrate enzyme kinetic models is thus of clear importance to the practicing biochemist. However, such models are infrequently explored in the undergraduate biochemistry laboratory. This deficiency suggests the need for well-defined, tractable, and economical methods for characterization of bisubstrate enzyme activity. The application of an established real-time colorimetric assay to investigate kinetic parameters of glutathione transfer by glutathione S-transferase is described. The reported method relies on commercially available materials and uses experimental conditions that are suited to application in the introductory biochemistry laboratory. Investigation of bisubstrate kinetic parameters was performed as part of a three-week laboratory experiment that emphasized protein extraction, purification, and characterization, leading to guided student development of bisubstrate enzyme kinetic models.