Zymography is an electrophoretic technique to measure the proteolytic activity of native enzymes on a nonreducing sodium dodecyl sulfate polyacrylamide gel. It is a simple and powerful tool to assess the amount of various proteases present in both physiological and pathological conditions. The concerned protease degrades the protein substrate that is incorporated with the gel and resolves during the incubation period. Staining with Coomassie brilliant blue (CBB) reveals the sites of proteolysis as clear white bands. The intensity and area of the bands are linearly related to the amount of protease present in the loaded sample. Matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9), also known as gelatinases, have the indigenous property to digest gelatin and several other protein molecules present in the extracellular matrix. Here, we describe the detailed protocols and methods of zymography, with a special emphasis on the determination of gelatinases present in conditioned culture media and tissue extracts.