Cancer immunotherapy using T cell receptor-engineered T cells (TCR-Ts) represents a promising treatment option. However, technologies for pre-clinical safety assessment are incomplete or inaccessible to most laboratories. Here, TCR-T off-target reactivity was assessed in five steps: (1) Mapping target amino acids necessary for TCR-T recognition, followed by (2) a computational search for, and (3) reactivity screening against, candidate cross-reactive peptides in the human proteome. Natural processing and presentation of recognized peptides was evaluated using (4) short mRNAs, and (5) full-length proteins. TCR-Ts were screened for recognition of unintended HLA alleles, and as proxy for off-target reactivity in vivo, a syngeneic, HLA-A*02:01-transgenic mouse model was used. Validation demonstrated importance of studying recognition of full-length candidate off-targets, and that the clinically applied 1G4 TCR has a hitherto unknown reactivity to unintended HLA alleles, relevant for patient selection. This widely applicable strategy should facilitate evaluation of candidate therapeutic TCRs and inform clinical decision-making.