Three kinds of determination methods of glutathione were introduced and evaluated.The results showed that the DTNB method had remarkable advantages in determining glutathione of yeast extraction.The influencing factors of determination were studied and the optimum determination conditions were acquired.This method showed good veracity and precision,and had no statistically significant difference compared with theoretical recovery rate.DTNB method can avoid the disturbance of cysteine and is a simple,fast and economic method.