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Diagnostics to accurately detect disease and monitor therapeutic response are essential for effective clinical management. Bioengineering, chemical biology, molecular biology, and computer science tools are converging to guide the design of diagnostics that leverage enzymatic activity to measure or produce biomarkers of disease. We review recent advances in the development of these 'activity-based diagnostics' (ABDx) and their application in infectious and noncommunicable diseases. We highlight efforts towards both molecular probes that respond to disease-specific catalytic activity to produce a diagnostic readout, as well as diagnostics that use enzymes as an engineered component of their sense-and-respond cascade. These technologies exemplify how integrating techniques from multiple disciplines with preclinical validation has enabled ABDx that may realize the goals of precision medicine.