HIV protease (PR) is identified as an important drug-screening target for the design of selective acquired immunodeficiency syndrome (AIDS) therapeutics. The SensoLyte® 490 HIV Protease Assay Kit uses an optimized FRET peptide substrate for a continuous measurement of HIV protease activities. This FRET-based fluorogenic substrate is derived from a natural processing site for HIV-1 PR. Incubation of the recombinant HIV-1 PR with this substrate results in specific cleavage and a time-dependent increase in fluorescence intensity that is linearly related to the extent of substrate hydrolysis. Because of its simplicity and precision in the determination of reaction rates required for kinetic analysis, this kit offers many advantages over the commonly used HPLC or electrophoresis-based assays for peptide substrate hydrolysis by retroviral PRs.