Gelatin zymography is an inexpensive and sensitive method to detect the relative gelatinase activity of the active and inactive matrix metalloproteinases (MMPs). The technique involves the separation of proteins through electrophoresis in a substrate copolymerized sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under a nonreducing condition, and the enzymes are renatured to detect the gelatinase activity. The MMPs are a group of zinc-dependent endopeptidases that degrade the components of the extracellular matrix, including the basement membrane. MMPs are linked to a variety of physiological and pathological processes and are considered potential therapeutic targets in the treatment of diseases. The gelatinases are a class of MMPs that includes gelatinase A, or MMP-2 (72 kDa), and gelatinase B, or MMP-9 (92 kDa). These MMPs are found to have proteolytic activity on native collagen types and break down structural proteins in the extracellular matrix. The activity of the MMPs is found after the gel staining as transparent bands against a dark-blue background. In this book chapter, we aim to describe the detailed procedure to detect the gelatinolytic activity of MMP-2 and MMP-9 in human hepatocellular carcinoma (HCC) tissues and cells.