Matrix metalloproteinases (MMPs) are proteolytic enzymes regulating the degradation of collagen as well as other proteins in the extracellular matrix (ECM), tissue repair, and remodeling. Validating MMPs as current drug targets can potentially establish the clinical relevance of their quantitative expression and distribution and open new therapeutic venues in tumor and metastasis. Zymography is a technique used for the identification of the proteolytic activity of MMPs in diverse biological samples. In gel diffusion zymography, analysis of the functional activity of the gelatinases (MMP-2 and MMP-9) by the digestion of gelatin as a substrate, added to agarose gel to assess the active forms of MMPs, is done. Here we describe a detailed experimental protocol to quantitate the gelatinase activity within the cell culture.