The invention provides a novel assay for NS2/3 cleavage detection, particularly, a novel assay for the detection of the NS3 cleavage product in the presence of uncleaved NS2/3 based on discrimination of the activity of the resulting NS3 protease product from the NS3 protease activity of the NS2/3 uncleaved protein. Certain reaction conditions allow this discrimination in situ without having to resort to physical separation of both proteases by decreasing the NS3 protease activity of the NS2/3 protein and increasing the NS3 protease activity of the resulting NS3 protease product, thereby producing a “signal window” upon which it is feasible to distinguish and measure NS3 protease activity produced from the cleavage of NS2/3.