A HCV protease substrate that incoporates an ester bond between residues P1 and P1. Due to ready transesterification of the scissile bond to the acyl-enzyme intermediate, this substrate shows very high kcat/Km values, enabling detection of activity with subnanomolar nonstructural protein 3 (NS3 protease) concentrations. It is widely used for the continuous assay of NS3 protease activity. Substrate cleavage is proportional to the enzyme concentration with a detection limit for NS3 between 1 nM and 250 pM. Upon cleavage of this substrate, fluorescence can be monitored at Abs/Em=355/500 nm. As an internally quenced fluorogenic substrate, it can be used to monitor HCV NS3 protease activity.