Hepatitis C virus (HCV) is the major causative agent of non-A non-B hepatitis, an important health problem with an estimated 50 million people infected worldwide. Among the possible targets for therapeutic intervention, the serine protease contained within the N-terminal region of nonstructural protein 3 (NS3 protease) is so far the best characterized.In vitrocharacterization of synthetic substrates based on all the natural cleavage sites (as well as a series of analogs) has consistently revealed poor kinetic parameters, making them unsuitable for sensitive high-throughput screening. To overcome these difficulties, we have recently developed depsipeptide substrates incorporating an ester bond between residues P 1 and P ' 1 . Due to ready transesterification of the scissile bond to the acyl-enzyme intermediate, these substrates showed very highk c a t /K m values, enabling detection of activity with subnanomolar NS3 concentrations. We have used the same principle to synthesize internally quenched depsipeptide fluorogenic substrates based on resonance energy transfer between the donor/acceptor couple 5-[(2'-aminoethyl)amino]naphthalene sulfonic acid/4-[[4'-(dimethylamino)phenyl]azo]benzoic acid, and developed a continuous assay for NS3 activity. Substrate cleavage is linear with enzyme concentration: depending on the conditions chosen, we estimated a detection limit for NS3 between 1 nmand 250 pm. The suitability of the assay for evaluation of inhibitors was established using as competitor a tridecapeptide corresponding to the natural NS4A/4B cleavage site; this gave an IC 5 0 of 30 μm, well in agreement with the previously foundK m value (40 μm).