Proteases are a family of naturally occurring enzymes in the body whose dysregulation has been implicated in numerous diseases and cancers. Their ability to selectively and catalytically turnover substrate adds both signal amplification and functionality as parameters for the detection of disease. This review will focus on the development of activity-based methodologies to characterize proteases, and in particular, the use of positional scanning, synthetic combinatorial libraries (PS-SCL's), and substrate activity screening (SAS) assays. The use of these approaches to better understand a protease's natural substrate will be discussed as well as the technologies that emerged.