Hydrolysis of insulin has been studied during storage of various preparations at different temperatures. Deamidation is the predominant degradation process in acid solution resulting in a desamido product. The current study examines whether the interaction of insulin with methyl-beta cyclodextrin (met beta CD) improves its stability. Hydrolysis of insulin was monitored by an HPLC assay with ultraviolet detection. The stability constant of insulin-met beta CD complex was calculated by Lineweaver-Burke linear equation. Furthermore, the complexation of insulin with met beta CD was characterized by (1)H NMR and Electrospray Mass Spectrometry (ESI-MS). Met beta CD had a stabilizing effect on insulin degradation according to the kinetic parameters, leading to a decreased chemical deterioration. Furthermore, the stability constant K(st) and the activation energy E(a) were calculated by fitting the kinetic results to Lineweaver-Burke and to Arrhenius linear equations, respectively. Finally, the complexation of insulin with met beta CD was characterized in aqueous media by (1)H NMR chemical shift displacements of assignable aromatic protons of specific amino acids upon the addition of the cyclodextrin, as well as by ESI-MS, since additional m/z peaks, which were attributed to insulin-met beta CD complex, were detected. It is concluded that addition of met beta CD resulted in a significant increase in the stability of complexed insulin compared with free insulin.