T cell antigen receptors (TCRs) on the surface of T cells bind specifically to particular peptide bound major histocompatibility complexes (pMHCs) presented on the surface of antigen presenting cells (APCs). This interaction is a key event in T cell antigen recognition and activation. Most studies have used surface plasmon resonance (SPR) to measure the in vitro binding kinetics of TCR-pMHC interactions in solution using purified proteins. However, these measurements are not physiologically precise, as both TCRs and pMHCs are membrane-associated molecules which are regulated by their cellular environments. Recently, single-molecule förster resonance energy transfer (FRET) and single-molecule mechanical assays were used to measure the in situ binding kinetics of TCR-pMHC interactions on the surface of live T cells. These studies have provided exciting insights into the biochemical basis of T cell antigen recognition and suggest that TCRs serially engage with a small number of antigens with very fast kinetics in order to maximize TCR signaling and sensitivity.