The aim of this study was to determine a cost-effective strategy for the diagnosis of hepatitis C virus (HCV) infection in clinical laboratories. Anti-HCV antibodies were sought in 3,014 consecutive unselected samples with two different enzyme-linked immunosorbent assays (ELISA). An immunoblot-based confirmatory assay (RIBA3.0) was performed in the samples with at least one ELISA positive or weakly positive. HCV RNA was evaluated using HCV polymerase chain reaction (PCR) in the samples with a weakly positive ELISA, discrepant results of the two ELISAs, or an indeterminate RIBA3.0 pattern. The two ELISAs gave concordant results in 2,957 (98.1%) of the 3,014 samples (negative in 87.9%, positive in 11.8%, and weakly positive in 0.3%), and discrepant results in 57 (1.9%). RIBA3.0 was positive in 338 of the 350 ELISA-positive samples (96.6%) and indeterminate in 12. Six of them were PCR-positive. Among the 8 weakly positive samples, 1 was RIBA3.0-positive, 6 were RIBA3.0-indeterminate, and 1 was RIBA3.0-negative; all were PCR-negative. Among the 57 samples with discrepant ELISA results, 4 were RIBA3.0-positive (none were PCR-positive), 22 were RIBA3.0-indeterminate (1 was PCR-positive), and 31 were RIBA3.0-negative (6 were PCR-positive). In these cases, the clinical context and PCR detection of HCV RNA allowed for definitive classification. In conclusion, one single ELISA determination is necessary for diagnosis of HCV infection in clinical laboratories, and confirmation of positive or weakly positive ELISAs with immunoblot-based confirmatory assays is no longer needed. HCV-RNA detection by PCR helps to resolve weakly positive or negative ELISA results when the clinical context is compatible with hepatitis C.