Proteolytic degradation of extracellular matrix (ECM) is a critical step during cell invasion and tissue transmigration that is required for many physiological and pathological processes. Cellular structures that mediate cell adhesion to, degradation of, and invasion into ECM are invadopodia of transformed and tumor cells and podosomes of normal monocytic, endothelial, and smooth muscle cells. Detecting the ability of the cell to form invadopodia and podosomes and to degrade ECM is required for studying the invasive capability of the cell. We have developed approximately 50 nm thick fluorescent gelatin matrices that provide a rapid, sensitive, and reliable in vitro system for detection of invadopodia and podosomes, and measurements of the extent of ECM degradation. In this chapter, we provide a detailed protocol for preparation of thin fluorescent gelatin matrices and for evaluation of the results from this degradation assay.