HIV protease (PR) is a key enzyme in the late phase of the HIV replication cycle. Proteolytic processing of two viral polyproteins (Gag and Gag-Pol) yields mature, infectious viral particles. Inactivation or inhibition of HIV PR renders the virus non-infectious, and HIV PR has thus become a prime therapeutic target. Major research endeavors by both academia and the pharmaceutical industry have led to ten HIV PR inhibitors currently used in antiretroviral therapy. However, the regulation, activation, and kinetics of HIV polyprotein processing and virion maturation are still not completely understood. The possibility to induce HIV polyprotein processing in a synchronized manner with an external stimulus would be an important step towards addressing these questions. Therefore, we designed, synthesized, and tested a potent, specific, photodegradable HIV PR inhibitor. The compound, an analogue of the tight-binding HIV PR inhibitor ritonavir (RTV), shows subnanomolar inhibitory activity in vitro. It contains a photolabile moiety which is released from the molecule upon irradiation with 405-nm light, reducing the inhibitory potential by four orders of magnitude. We determined the structure of the PR-inhibitor complex, analyzed its photolytic products, and showed that the enzymatic activity of inhibited HIV PR could be fully restored upon inhibitor photolysis. We also demonstrated that polyprotein processing of immature HIV containing the inhibitor could be triggered by light. An analogous approach may be used for photocaging of other biologically relevant proteases and for analysis of their function in situ.