Human adenovirus proteinase (AVP), the first member of a new class of cysteine proteinases, is required for the synthesis of infectious virus. As such, it is an attractive target for proteinase inhibitors that act as antiviral agents. However, before potential inhibitors can be screened, a quick, sensitive, and quantitative assay for the enzyme is required. Here, methods for purification of a recombinant AVP expressed in Escherichia coli are presented and a fluorogenic substrate is designed, synthesized, and purified and then used in the development of a quick, sensitive, and quantitative assay for the enzyme. The reporting group in the substrate is Rhodamine 110, possibly the most detectable compound known. The substrate contains the proteinase consensus cleavage sequence (Leu-Arg-Gly-Gly). The synthesis and purification of (Leu-Arg-Gly-Gly-NH)2-Rhodamine is described. It is then used to develop assays with AVP and its various cofactors. The resultant assays are quite sensitive; enzyme activity at low nanomolar concentrations can readily be detected.