Hepatitis C virus (HCV) NS3/4A (non-structural 3 and 4 B) protease plays a key role in the processing of polyprotein precursor and it becomes an attractive target for antiviral drug discovery. We developed a cell-based assay for monitoring of the NS3/4A protease activity in mammalian cells that is an important step in screening of specific drugs against the protease. The recombinant caspase 3 (rCasp3) was used as the specific substrate for NS3/4A protease. The endogenous cleavage sites in the procaspase 3 molecule were substituted by decapeptides specific for NS3/4A protease. The activation of rCasp3 depended on its specific cleavage by NS3/4A protease and resulted in an apoptosis of stable cells expressing the protease. The difference in cell viability between the cells expressing NS3/4A protease transfected with rCasp3 and the counterparts pretreated with NS3/4A protease inhibitors could be estimated by a spectrophotometry based on 3-(4,5-dimethylthioazol- 2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) staining of cells in microplates. Thus, we developed a simple and cost-effective colorimetric assay for evaluating NS3/4A protease activity enabling the screening of candidate NS3/4A protease inhibitors.