Abstract: Objective To optimize the derivatization conditions of glutathione with N-ethylmaleimide (NEM) in whole blood to achieve the complete derivatization. Methods Sixty adults with 29 males and 31 females (23-79 years) in the Department of Clinical Laboratory of Chinese PLA General Hospital from April 2016 to June 2016 were enrolled in this study.Venous blood of 2 ml was collected respectively to determine the optimal derivatization reaction condition and the normal glutathione (GSH) concentration value.The effects of temperature, pH and reaction time on the derivatization process were analyzed by liquid chromatographytandem mass spectrometry (LC-MS/MS).Group A: Samples were allowed to react at room temperature(25℃) for 10, 30, 60 min, respectively.Group B: Samples were heated at 50, 60, 70, 80, 90℃ in a water bath for 10 min, respectively.Group C: The pH was adjusted with Tris-Cl buffer (pH=8) and heated at 25, 70, 80, 90℃ in a water bath for 10 min, respectively.Group D: After derivatization, samples were heated at 25, 70, 80, 90℃ in a water bath for 10 min and then add precipitant reagent. Results The 25℃ subgroup of group D showed the lowest relative rate of non-derivatized GSH response compared with other groups (P＜ 0.05, respectively).The concentrations of GSH-NEM and oxidized glutathione (GSSH) in whole blood of 60 healthy adults were(1 471.61±269.43) µmol/L and (0.84±0.38) µmol/L with the detection method of group D. Conclusion When the derivatization reagent NEM and the precipitant reagent 5- sulfosalicylic acid (SSA) are added respectively, a thorough derivatization reaction can be achieved without additional adjustment of pH and temperature.