Fluorogenic sugar derivatives are useful substrates for the detection of glycosidase activity within a native polyacrylamide gel. When an enzyme cleaves the glycosidic bond, a fluorescent compound is released at the protein’s location, forming a band visible under UV light. This method can be used to identify proteins with glycosidase activity within a mixture, evaluate the effect of mutations on enzyme activity, and even provide information about the quaternary structure of a glycosidase. Here, we present a protocol for glycosidase zymography and demonstrate its utility as a tool to probe the active oligomeric form of a novel bacterial β-galactosidase.