A simple flow injection spectrophotometric method is reported for the determination of cysteine, N-acetyl cysteine and glutathione based on the reduction of Fe(III)/ferricyanide, the in situ reduced ions are reacted with unreduced portion of ferricyanide/Fe(III) to form soluble Prussian blue, which is monitored at 735 nm. The calibration graphs are linear in the concentration ranges of (1―100)×10–6 mol/L for cysteine and N-acetyl cysteine, and (1―50)×10–6 mol/L for glutathione. The relative standard deviations of 1.8%, 2.5% and 1.9% were found for eleven replicate analyses of 5×10–6 mol/L cysteine, N-acetyl cysteine and glutathione. The limits of detection(3σ blank) at 5×10–7 mol/L for cysteine, and 3×10–7 mol/L for N-acetyl cysteine and glutathione were obtained. The proposed method allowed 60 injections/h. The effects of common substances present in pharmaceuticals and human physiological fluids were examined. The method was applied to determining cysteine in pharmaceutical formulations with the recoveries in a range of 97% to 106% and the results obtained are agreed well with labeled values.