In the present study, new putative epitopes located in structural (E2) and non-structural (NS3) proteins of GBVC / HGV were identified by computer-aided prediction of antigenicity and synthesized in solid-phase, following an FmoctBut strategy, for their use in immunoassays. The corresponding synthetic peptides were used as antigens in ELISA assays and in real-time biospecific interaction measurements. This last approach allowed direct detection of GBVC / HGV-specific antibodies in human sera. Good correlations were obtained between the biospecific interaction analysis and the ELISA. To verify the performance of these new assays in comparison to the existing recombinant E2 protein commercial test, antibodies to synthetic peptides were searched for in different panels of serum samples. The main conclusion of this work is the usefulness of E2 peptides in the detection of antibodies. Moreover, the NS3 peptide could be exploited to improve the sensitivity of the currently available test. Our results offer a new approach to develop new diagnostic peptide based biosensors for serodiagnosis of GBV-C / HGV infection.