Abstract: To explore the fragmentation reactions of non-covalent complex, the complex of glutathione(GSH) and amino acids were chosen to investigate the main factors affecting the fragmentation process. The secondary tandem mass spectrometry(MS²) results indicated that collision gas thickness(CGT) and collision energy are two important conditions affecting the fragmentation pathway of glutathione complex. When the CGT maintained at 50×10¹³ molecules/cm², increasing collision energy from 5 eV to 80 eV, the dissociation of His-GSH complex took place, leading to the formation of His+H⁺ and GSH+H⁺. However, it could be seen that no further dissociation of GSH occurred， and the fragmentation of noncovalent bond was the main pathway of the complex. When CGT rose up to 75×10¹³ molecules/cm², the covalent bond also began to dissociate, leading to the formation of y₂ and b₂ for glutathione. Such phenomena could be also observed in Lys-GSH complex. To confirm the fragment ion y₂ of glutathione, MS³ was also performed and y₁ was detected.