The degradation of the extracellular matrix during development and in disease is thought to result from the combined action of several proteolytic enzyme systems, including the matrix metalloproteinases (MMPs), serine proteinases, and cysteine proteinases. The majority of the soluble MMPs are synthesized as proenzymes which require extracellular activation in order to gain proteolytic activity and the analysis of their activation mechanism is a prerequisite for understanding MMP-mediated proteolysis.The emphasis of this chapter is the provision of the experimental tools to study MMP activation in vitro and in cellular model systems. Hence, we use the activation of procollagenase-3 (proMMP-13) and progelatinase A (proMMP-2) as examples of the methods used.