Zymography is a powerful technique that can be exploited to specifically assess the relative expression levels of a group of proteolytic enzymes termed matrix metalloproteinases (MMPs) through their catalytic activity. It is further used to monitor the ratios status of activated over latent MMP forms that provide more accurate insights in their physiological roles in regulating the degradation of the extracellular matrix. Clinical tissue biopsies, in vitro primary cell culture lysates, or media conditioned by tumor-derived preclinical in vitro cell cultures can be used by researchers to assess by zymography clinical treatment efficacy or pharmacological impact of drugs in development on MMPs level. As increases in MMPs protein levels do not necessarily correlate with increased enzymatic activity, assessing MMPs in clinical tissue samples or from preclinical cell models using zymography is the best indicator of the impact of a given therapy on the activation status of these enzymes, or the impact on an invasive molecular phenotype in the case of tumor biopsies. In this chapter, the proteolytic activity of MMP-2 and MMP-9, two gelatinases, is detected as unstained clear digested bands against a stained gelatin background in polyacrylamide gels. Zymography’s strengths are its cost-effectiveness, rapidity, and adaptability since it can be used with a relatively small amount of starting material to assess the activation status and proteolytic activity of other MMPs types when used in gel with their specific substrates.