Hepatitis C virus (HCV) is one cause of non-A non-B hepatitis with 1 -2 % of the worldwide population estimated as carriers: a high percentage of asymptotic carriers get chronic hepatitis that may lead to cirrhosis and hepatocellular carcinoma. The virus belongs to Flaviviridae family, and the genomic arrangement is 5’-C-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B-3’, where C-E1-E2-p7 encodes structural proteins and NS2-NS3-NS4A-NS4B-NS5A-NS5B encodes non-structural proteins. The product translated from 5’-terminal one-third of NS3 has serine protease activity, which is responsible for the cleavage of nonstructural proteins enable them to mature. NS3 protease is suggested to play a role in pathogenesis, given its transformation of some cell lines and interaction with tumor suppression protein, p53. Structural analysis by X-ray crystallography showed that NS3 protease has a trypsin-like arrangement of an active triad, His-57, Asp-81 and Ser-139, located at the central groove between 2 β-barrels. The protease is considered a good target of anti-HCV agents, and many attempts have been made to find inhibitors of the enzyme. To develop of inhibitors as candidates for antivirus agents, a rapid sensitive assay for the enzyme is required to screen a large number of compounds. We report here our approach to developing a series of high-throughput assays including HPLC, ELISA and colorimetric detection performed on a multiwell plate; results of screening a chemical library of 2,000 compounds and a group of natural occurring products.