HCV is a potential cause of viral hepatitis, which leads to blood-borne chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. Anti-HCV antibody detection assays detect HCV infection after approximately 70 days. HCV core antigen can be detected much earlier than anti-HCV antibodies. However, it disappears soon after the appearance of anti-HCV antibodies. Thus, there is a need for a rapid assay for simultaneous detection of HCV core antigen and anti-HCV antibodies for early diagnosis of HCV infection. A rapid diagnostic assay (HCV Ag-Ab Combo) for simultaneous detection of HCV core antigen and anti-HCV antibodies for early diagnosis of HCV infection was developed. HCV Ag-Ab Combo was evaluated in order to determine its potential for detection of HCV infection earlier than anti-HCV antibody detection assays. We compared the sensitivity of the newly developed assay with anti-HCV antibody detection assays (ELISA [HCV Ab ELISA] and rapid test [HCV Ab Rapid]) and HCV core antigen/anti-HCV antibody detection ELISA (HCV Ag-Ab ELISA). This study included 11 samples that were found positive in HCV RNA detection and HCV Ag-Ab ELISA but negative in HCV antibody detection assays (HCV Ab ELISA and rapid), 10 samples that were found positive in HCV Ag-Ab ELISA and HCV Ab ELISA but negative in HCV Ab Rapid, 69 samples that were found positive in HCV Ag-Ab ELISA, HCV Ab ELISA, and HCV Ab Rapid, and 509 samples that were found negative in HCV Ag-Ab ELISA, HCV Ab ELISA, and HCV Ab Rapid. Three seroconversion panels, PHV 913, PHV 911 (M) and PHV904-00-1.0, and a HCV RNA genotype qualification panel (2400-0182) acquired from Seracare Life Sciences (USA) were also tested. HCV Ag-Ab Combo showed a combined sensitivity and specificity of 100% when tested with 90 samples that were positive for HCV by HCV Ag-Ab ELISA and 509 HCV-negative samples. Its positive predictive value (PPV) and negative predictive value (NPV) were found to be 100%. It detected HCV infection approximately 7 to 12 days earlier than the HCV Ab detection assays, and its performance was not affected when testing different genotypes of HCV. HCV Ag-Ab Combo did not detect HCV infection as early as HCV RNA or HCV Ag-Ab ELISA. HCV Ag-Ab Combo provided a significant improvement for the early detection of HCV infection during the preseroconversion phase when compared with anti-HCV antibody detection assays. It could be a useful screening assay, and an alternative to HCV RNA detection or HCV Ag-Ab ELISA when nucleic acid technologies cannot be implemented.