Proteases are important targets for the treatment of human disease. Several protease inhibitors have failed in clinical trials due to a lack of in vivo specificity, indicating the need for studies of protease function and inhibition in complex, disease-related models. The tight post-translational regulation of protease activity complicates protease analysis by traditional proteomics methods. Activity-based protein profiling is a powerful technique that can resolve this issue. It uses small-molecule tools—activity-based probes—to label and analyze active enzymes in lysates, cells, and whole animals. Over the last twelve years, a wide variety of protease activity-based probes have been developed. These synthetic efforts have enabled techniques ranging from real-time in vivo imaging of protease activity to high-throughput screening of uncharacterized proteases. This Review introduces the general principles of activity-based protein profiling and describes the recent advancements in probe design and analysis techniques, which have increased the knowledge of protease biology and will aid future protease drug discovery.