The roles of proteases in cancer are now known to be much broader than simply degradation of extracellular matrix during tumor invasion and metastasis. Furthermore, proteases from tumor-associated cells (e.g., fibroblasts, inflammatory cells, endothelial cells) as well as tumor cells are recognized to contribute to pathways critical to neoplastic progression. Although elevated expression (transcripts and proteins) of proteases, and in some cases protease inhibitors, has been documented in many tumors, techniques to assess functional roles for proteases require that we measure protease activity and inhibition of that activity rather than levels of proteases, activators, and inhibitors. Novel techniques for functional imaging of protease activity, both in vitro and in vivo, are being developed as are imaging probes that will allow us to determine protease activity and in some cases to discriminate among protease activities. These should be useful clinically as surrogate endpoints for therapies that alter protease activities.