Blood glutathione (exists in both reduced and oxidized states) was firstly stacked and separated as a cation using acetonitrile–salt stacking method by capillary electrophoresis. Blood samples were treated with 200 mM phosphoric acid and acetonitrile at a final volume ratio of 2 : 1 : 1 and supernates were directly assayed after centrifugation (15 000 g, 15 min). Several factors influencing the separation and stacking were systematically investigated and optimized. Under the selected conditions, blood glutathione can be detected within 35 min with satisfactory limit of detection (0.1–0.3 μM), relative standard deviation (<1.0% for migration time; <2.0% for peak area) and linearity range (0.3–640 μM), which proves that the proposed method is suitable for routine glutathione determination in blood samples. [ABSTRACT FROM AUTHOR]