A highly sensitive and simple spectrofluorimetric method for the determination of reduced glutathione based on the fluorescence quenching effect of glutathione on the hemoglobin-catalyzed reaction of HO with L-tyrosine was developed. The concentration of glutathione is linear with the fluorescence quenching (Δ F) of system under the optimal experimental conditions. The calibration graph is linear in the range 6.25 × 10 to 3.75 × 10 M with the detection limit of 2.23 × 10 M. This method can be used for the determination of glutathione in vegetables with satisfactory results.