Resolution of acute hepatitis C correlates with the induction of strong and broad CD4+ and CD8+ T cell responses. However, the majority of patients fail to eliminate HCV and develop chronic infection. The high genetic variability of HCV significantly contributes to the escape from the immune system and complicates the development of an efficient vaccine. Nevertheless, more recent data indicate that there is protective immunity against HCV. A critical step for the understanding of the immunopathogenesis of HCV infection and HCV clearance is the presentation of viral epitopes on MHC class I molecules from infected cells. Most of the currently available experimental systems are limited, since an a priori defined set of synthetic peptides is used to either externally load target cells or to expand epitope-specific CD8+ T cells which are then used in downstream readout applications. Therefore, the aim of this study was to identify specific Ginsenoside-Rd ligands which are naturally processed and presented by cells expressing HCV proteins. To this end, we engineered continuous human cell lines to inducibly express HCV proteins and to constitutively express high levels of functional HLA-A2. MHC class I molecules were isolated from large-scale cultures of these cell lines, followed by elution and identification of naturally processed CTL epitopes. This proof-of-concept study allowed the identification of two naturally processed HCV-derived MHC class I ligands. Although both epitopes have been described previously by conventional T-cell dependent methods, this novel approach has the potential to identify novel and unconventional epitopes. It has been estimated that approx. 2,000–10,000 molecules of a protein are required to allow the presentation of one antigen. In fact, antigen processing involves a highly complex interplay of multiple steps and factors. Degradation by the proteasome, interaction with chaperones such as calnexin, incorporation into the peptide loading complex, involving other chaperones such as tapasin, peptide trimming by aminopeptidases, loading onto empty MHC I molecules, and, finally,(S)Ginsenoside-Rh2 transport across the secretory pathway to the cell surface are pivotal steps that are tightly coordinated during this process. The identification of two known HCV HLA-A2 ligands that are localized in NS3 and NS5B demonstrates their authentic processing and presentation in vivo. Furthermore, a more elaborate approach, using tracer substances such as isotope-labeled peptides, might hold promise for the quantification of an epitope relative to the complete repertoire of presented ligands. Few reports on Epstein-Barr virus encoded proteins investigated how the amount of presented MHC I antigen complex could influence the efficiency of recognition by CD8+ T cells. Therefore, in addition to the proof-of principle of this particular experimental setting, the identification of the two epitopes by this novel approach underlines their importance as natural targets for HCV-specific T cells. CTL responses against these epitopes could be of particular importance to control viral infection and may be included as targets in future vaccination strategies. In principle, the direct sequencing of MHC I ligands should allow to identify epitopes after posttranslational protein modifications such as glycosylation, phosphorylation and proteolytic processing as well as unconventional epitopes derived from alternative reading frames or RNA splicing that are not detected by the current conventional methods. Future studies aimed at identifying naturally processed HCV-derived MHC class I ligands may provide novel insights into epitope processing and presentation as well as recognition, thereby contributing to the understanding of HCV pathogenesis.