Gliomas are tumors with low mutational burden with the majority of them being resistant to checkpoint inhibition due to few immunogenic antigens. Multicenter vaccine trials targeting personalized neoantigens in gliomas demonstrated feasibility and illustrated the challenges of retrieving neoepitope-specific T cells based on the prediction of immunogenic neoepitopes. Here we took an entirely different T cell-centric approach and established a single cell sequencing-based high-throughput T cell receptor (TCR) retrieval platform, exploiting the therapeutic potential of spontaneous intratumoral T cell clonotypes for the development of adoptive cell therapy. We conducted direct ex vivo TCR single cell sequencing from freshly sorted human glioma-infiltrating T cell samples. High fidelity PCR was established to clone TCRs from single cell libraries directly into episomal expression vectors further optimized for T cell therapy. In parallel to standard therapy, patient-derived xenografts were developed and characterized. Tumor-reactivity of retrieved TCRs was demonstrated against patient-derived cell lines. Collectively, we provide a novel sequencing-based platform for high-throughput identification and validation of endogenous glioma-targeting TCRs and demonstrate their therapeutic applicability.