Proteases are important drug targets; the first drugs to reach the market were antihypertensives and antivirals. Designing HTS assays for these targets requires thorough understanding of the biochemistry and biology of these enzymes as well as various assay formats. In this chapter, the authors present basic properties of protease enzymes, sensitive fluorescent assay formats compatible with HTS, both in homogeneous and separation-based approaches. Sections on hit selection and data analysis concepts for identifying inhibitors are also included with an extensive set of literature references.