Patient-Derived Tumor Organoids (PDTOs) have emerged as physiologically relevant systems, retaining genomic and phenotypic features of patient tumors and enabling drug response prediction. Here, we established a patient-specific pipeline to evaluate precision immunotherapy in Renal Cell Carcinoma (RCC) and Bladder Cancer (BC), two highly immunogenic malignancies. Tumor and adjacent benign tissues were used to generate PDTOs and Patient-Derived Cells (PDCs) for immunopeptidome profiling. Using our in-house microfluidic platform, PeptiCHIP for immunoaffinity purification followed by Ligandomics LC/MS analysis, we identified tumor-associated peptides presented by HLA-I molecules as potential targets for T cell–based immunotherapies. Their immunogenicity was later tested with peptide-expanded HLA-matched PBMCs, leading to the identification of peptides capable of eliciting antigen-specific CD8+ T cell activation and cytotoxicity against the patient’s tumor.