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Fluorogenic Human CMV Protease Substrate, the FRET substrate, is used to develop a fluorescence-based detection method for human cytomegalovirus proteinase. It specifically cleaves on the Ala-Ser bond, thereby removing the C-terminal peptide-Edans fragment from the proximity quenching effect of DABCYL groups. This represents the first fluorescence-based assay of the herpes virus protease and allows characterization of potential inhibitors.