Recent reports have demonstrated the potential of monocyclic beta-lactam derivatives as inhibitors of human cytomegalovirus (HCMV) protease. Investigation of the mechanism of inhibition by NMR and mass spectrometry has revealed the presence of an acylenzyme intermediate suggesting that beta-lactams are hydrolyzed by the enzyme and cause inhibition by competing with substrate. The potential of a fluorogenic beta-lactam derivative for convenient kinetic characterization of this mechanism has been evaluated using 4S-(4methylumbelliferone)-3R-methylazetidin-2-one-1-carboxylic acid (4-methylpyridyl) amide (1). Upon acylation of the enzyme, the fluorescent umbelliferone moiety is released, allowing for continuous monitoring of the hydrolytic process. Examination of a series of progress curves by numerical analysis has provided valuable information on acylation and deacylation rates which relate to the IC50 values observed for beta-lactams. More importantly the potential of compound 1 as an active site titrating agent for HCMV protease has been exploited, and a simple protocol for rapid determination of active enzyme is described. The data are consistent with the HCMV protease dimer being composed of two functional active sites. This titrating agent represents an important tool that should significantly facilitate the characterization of this novel enzyme.