In many diseases, protease expressions are found deregulated when compared with them at the healthy states. The unique ability to hydrolyze protein amide bonds has made those deregulated proteases attractive biological triggers in drug development. Proteolysis has been widely applied in pro-drug design to achieve favorable pharmacokinetics. Controlled drug delivery systems are also reported by incorporating protease-sensitive motifs onto bio-, inorganic-, or organic- materials. In addition, protease responsive molecular probes are developed for in vitro bioanalysis and in vivo diagnostic imaging. This review focuses on various proteolysis-dependent approaches to drug delivery, therapy, and imaging. References are selected to illustrate the concepts and demonstrate the potentials of these enzyme-responsive strategies.