Proteases are enzymes that cleave peptide bonds within proteins, and this function is critical for cellular processes ranging from digestion and regulation of protein homeostasis to apoptosis and immune function. Aberrant protease activity is a hallmark of many diseases including cancer, inflammation, neurodegenerative diseases, and pain. Proteases are thus attractive as targets for both the diagnosis and treatment of these conditions. As protease activity is tightly controlled at the post-translational level, measures of total protein expression rarely reflect the pool of active, functional protease. We have developed a suite of peptide-based chemical tools called activity-based probes (ABPs) that can measure the specific activity of diverse cysteine and serine proteases. Our laboratory applies these tools to measure protease activity in whole animals by non-invasive imaging, in tissues by ex vivo imaging, and at the cellular level by confocal microscopy and flow cytometry. The major innovation of ABPs is that they covalently modify their protease targets, allowing for careful validation by in-gel fluorescence, immunoprecipitation or activity-based proteomics. This talk will highlight the value and versatility of ABPs, particularly with respect to their applications as pharmacological tools to support drug discovery (inhibitor development, monitoring of target engagement, target validation, and application to fluorescence-guided surgeries).