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Aim: To study the non-covalent interaction between glutathione and common amino acids. Methods: A stoichiometry of glutathione and common amino acids were mixed to reach the equilibrium, and then the mixed solution was investigated by electrospray ionization mass spectrometry (ESI-MS). The binding of the com-plexes was further examined by collision-induced dissociation (CID) in a tandem mass spectrometer as well as UV spectroscopy. To avoid distinct ionization effi-ciency discrepancy and signal suppression in the ESI-MS measurements, the interaction between glutathione (GSH) and glutamate (Glu) was quantitatively evaluated. The total concentrations and series of m/z of peak intensities for glu-tathione and amino acids could be achieved, respectively. Due to the existence of some oligomeric species arising from glutathione or amino acids, an improved calculation formula was proposed to calculate the dissociation constants of glu-tathione binding to amino acids. Results: The ESI mass spectra revealed that glutathione could interact easily with Met, Phe, Tyr, Ser, or lie to form non-cova-lent complexes. The binding of the complexes was further confirmed by CID experiments in a tandem mass spectrometer as well as UV spectroscopy. Moreover, an improved calculation formula was successfully applied to determine the disso-ciation constants of glutathione binding to Glu, His, or Gln. Finally, a possible formation mechanism for the complexes of glutathione with amino acids was proposed. Conclusion: The reduced polypeptide y-glutathione can interact with each of 8 common amino acids, including Glu, His, and Gin to form non-covalent complexes with different affinity.