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A novel two-step bioinformatics strategy was applied for identification of signatures with therapeutic implications in hepatitis-associated HCC. Transcriptional profiles from HBV- and HCV-associated HCC samples were compared with normal liver tissue controls. Resulting HCC modulated genes were subsequently compared with different normal tissue samples. Two related signatures were identified, namely “HCC-associated” and “HCC-specific.” Expression data were validated by RNA-Seq analysis carried out on unrelated HCC samples and protein expression was confirmed according to the publicly available database at http://www.proteinatlas.org/. Among all, only two proteins (namely AKR1B10 and IGF2BP3) were found strictly HCC-specific with no expression in 18/20 normal tissues. Target peptides for vaccine design were predicted for both proteins associated with the most prevalent HLA-class I and II alleles. The described novel strategy showed to be feasible for identification of HCC-specific proteins as highly potential target for HCC immunotherapy (Petrizzo et al., Scientific Reports, in press). Citation Format: Annacarmen Petrizzo, Francesca P. Caruso, Maria Tagliamonte, Maria Lina Tornesello, Michele Ceccarelli, Valerio Costa, Marianna Aprile, Roberta Esposito, Gennaro Ciliberto, Franco M. Buonaguro, Luigi Buonaguro. Identification and validation of HCC-specific gene transcriptional signature for tumor antigen discovery [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A046.