Proteases, also known as peptidases or proteinases, are enzymes that break down peptide bonds of proteins. It is essential for number of physiological processes like digestion, immune response, blood clotting, and cell regulation. Different versions of the same protease enzyme, or isoforms, result from post-translational modifications, alternative splicing, or genetic differences. These isoforms add another level of complexity and specificity to protease action with different cellular localizations, substrate specificities, and regulatory characteristics. Substrates like caesin and gelatin are coplymerized with the gel. Gelatin zymography is used for examining protease activity, especially for different isoforms. This reliable method evaluates the activity of proteases, particularly serine and matrix metalloproteinases (MMPs). It involves the electrophoretic separation of proteases in polyacrylamide gels embedded with gelatin or casein as a substrate. After electrophoresis, the gel is incubated for the gelatin degradation by proteases, resulting in clear bands on background of the stained gel where proteolytic activity has occurred. This technique is useful for studying the disease condition such as cancer and inflammatory disorders by revealing the activity of proteases-specific MMP activities. Recent improvements in cost-effectiveness and detection sensitivity have enhanced the utility of zymography. These advancements have improved our ability to assess and interpret protease activity in various biological contexts, highlighting the significance of zymography in both environmental research and clinical diagnostics. In this chapter, the basic methods for the protease zymography for instance gelatin and casein have been discussed with the main safety precautions and troubleshooting techniques required for the gelatin zymography.