The measurement of glutathione is a challenging task in that, during sample manipulation, a large percentage of this compound can be artificially oxidized. Here a high-performance liquid chromatography (HPLC) method to measure reduced glutathione in blood, based on the analysis of its conjugate with N-ethylmaleimide, has been developed and validated. The method is linear in the concentration range 0.1-2 mM, and the lower limit of quantification is 0.05 mM. Blood samples treated with N-ethylmaleimide are stable at -20°C for 90 days. This method has simplicity and rapidity as its main advantages; therefore, it is particularly suitable for large-scale clinical studies.