Imaging of enzyme activity in living subjects promises many applications in both basic and translational researches from helping elucidate the enzyme function and mechanism in biology to better disease detection and monitoring, but the complexity and dynamics of enzymatic reactions in living systems present unique challenges for probe design. This critical review examines the approaches in recent literature to in vivo imaging of the activity of a variety of enzyme targets with an emphasis on the chemical perspective of probe design, structure and function. Strategies for designing enzyme-activated probes based on a variety of molecular scaffolds including small molecules, organic and inorganic nanoparticles, and genetically encoded proteins for commonly used molecular imaging modalities—whole body optical (fluorescence, bioluminescence) imaging, magnetic resonance imaging, and radionuclide-based tomographic imaging, are critically evaluated. Recent advances in combining multiple modalities to imaging enzyme activity in living subjects are also highlighted (255 references).