Viral oncogenes E6 and E7 are ideal targets for therapeutic vaccines against human papillomavirus (HPV)-associated cervical cancer (CC). T cell-mediated immunity plays a crucial role in the clearance of HPV infection and regression of intraepithelial neoplasia. Current strategies for therapeutic vaccine development predominantly depend on immunoinformatic predictions of human leukocyte antigen (HLA)-restricted cytotoxic T lymphocyte (CTL) epitopes. Three T-cell epitope prediction programs were used to identify HPV16 E6 and E7 epitopes restricted to HLA-A*02:01. Subsequently, in silico evaluations were performed using five bioinformatic databases and computational servers. The binding affinities of these peptides to HLA-A2 molecules were experimentally validated using a T2 cell-binding assay. The effectiveness of the vaccine developed by combining peptides and CpG-containing oligonucleotide (CpG-ODN) was validated by inducing the generation of CTLs ex vivo, and its immunogenicity was verified in HLA-A*02:01/H-2Dd (AAD) transgenic mice. Eight HLA-A*02:01-restricted candidate peptides were preliminarily identified, and all candidate peptides demonstrated binding capabilities to HLA-A2 molecules. Using the integrated approach, four high-affinity peptides were successfully identified. Notably, these peptides also exhibited the potential to induce dendritic cell maturation, enhance the activation and proliferation of CD8+T cells, and elicit potent antigen-specific CTL responses against tumor cells. These findings support the potential application of the selected peptides in CTL-based immunotherapy for HPV-driven malignancies. Furthermore, the described peptide-screening platform proved to be an effective strategy for the rational design of candidate antigens for HPV therapeutic vaccines.Copyright © 2025 Wolters Kluwer Health, Inc. All rights reserved.