Gelatin zymography is a simple semiquantitative technique for assaying gelatinases in various biological samples. It is particularly feasible to measure two gelatin-degrading matrix metalloproteinases, MMP-2 (gelatinase A) and MMP-9 (gelatinase B). The use of SDS-polyacrylamide gels impregnated with gelatin provides a reliable way to detect and quantify the type, amount, and activation level of gelatinases in biological samples (cell lysates, tissue extracts, and biological fluids). This method can be used to investigate the expression profile and activation level of gelatinase in cancer cells or tissues and to determine how these changes are related to cancer progression. However, it is crucial to understand the principle and limitation of gelatin zymography before interpreting the experimental data. Gelatin zymography is a powerful method for assaying gelatinases in cancer research if some precautions are taken.