Proteases serve important functions for eukaryotes and bacteria, such as processing other proteins to activate them or degrading other proteins to create smaller peptides or eliminate malformed proteins. Proteases in pathogenic bacteria can also act as virulence factors by virtue of degradation of host proteins, activating other proteins that then enhance virulence, or eliciting bystander inflammatory damage through stimulation of the host immune response, to name a few functions. Zymography of a bacterial protein preparation is a powerful tool for visualizing and identifying the protease profile of a given bacterial strain. A sodium dodecyl sulfate (SDS)-polyacrylamide gel is impregnated with a protease substrate such as gelatin. Following electrophoresis, removal of SDS, incubation in buffer favoring protease activity, and staining, the substrate within the gel will retain the stain while clear bands will emerge where the protease activity degrades the gelatin. This method can be modified with different substrates, buffers, and other variables as detailed in the notes.