This hexapeptide FRET substrate is derived from the p24/p15 cleavage site of the viral gag-pol poly-protein. A simple, continuous fluorometric assay for HIV protease has been developed, which allows the screening of potential HIV protease inhibitors. Values for Km (2.1 μM) and kcat (7.4 s⁻¹) were calculated from steady-state data at a low concentration of the enzyme. Excitation at 280 nm, emission at > 435 nm.