A single T cell receptor (TCR) can recognise up to one million different peptide ligands with varying affinities. We have developed a novel high-throughput Surface Plasmon Resonance (SPR) method to measure the affinity of hundreds of TCR/pMHC interactions in a single experiment. Using this method, we demonstrate that T activation correlates with ligand affinity. Furthermore, we have shown that modifying co-signalling receptors can selectively abolish T cell activation against low-affinity peptide. Overall, these findings demonstrate that T cells can be engineered to be less cross-reactive, which has important implications for enhancing the safety of adoptive T cell therapies.