A fluorescence polarization assay was designed to measure proteolytic cleavage of a specific peptide substrate for human cytomegalovirus protease. The peptide substrate was derivatized by biotinylation of a gamma-aminobutyric acid-modified amino-terminus and labeled with 5-(4,6-dichlorotriazinyl)aminofluorescein at the carboxy-terminus. Incubation of this substrate with recombinant human cytomegalovirus protease and subsequent addition of egg white avidin produced a polarization signal that was proportional to the relative amounts of cleaved and uncleaved substrate. The uncleaved substrate produced a high polarization value upon binding to avidin, whereas the cleaved, low-molecular-weight fluorescently tagged peptide that cannot bind to avidin produced a low polarization value. The inhibitory activity of a 3,4-dichloroisocoumarin against the protease was evaluated by comparing the change in polarization with a noninhibited control. The fluorescence polarization protease assay does not suffer from interference due to the presence of absorptive interferants making this a convenient, homogenous assay for high throughput screening.