Abstract: To investigate the non-covalent interaction between glutathione and D-amino acids, stoichiometric reduced glutathione (GSH) and three D-amino acids, including D-phenylalanine (D-Phe), D-histidine (D-His), and D-glutamine (D-Gln), were mixed respectively, and then incubated at room temperature for 1 h to reach equilibrium. The electrospray ionization mass spectrometry (ESI-MS) results indicated that glutathione and three D-amino acids could formnon-covalent complexes in physiological pH conditions, respectively. The binding of glutathione to D-amino acid was further confirmed by collision-induced dissociation (CID) in a tandem mass spectrometer. Additionally, the complexes exhibited different features and properties from the reactants in UV spectroscopy, which also confirmed the results of ESI-MS. To avoid distinct ionization efficiency discrepancy and signal suppression in ESI-MS measurements, the interaction between glutathione (GSH) and D-glutamine (Gln) was quantitatively evaluated. A series of samples with different initial concentrations of glutathione and D-amino acids were mixed, and then series of peak intensities for different species in the mixture were achieved by ESI-MS. The dissociation constants of three complexes formed by glutathione and D-amino acids were calculated. The calculation results revealed that the reduced tripeptide γ-glutathione could interact with D-amino acids to formnon-covalent complexes with different affinities, the stabilities of the three complexes increased gradually according to the order of D-glutamine, D-phenylalanine, and D-histidine.