A rapid, sensitive, and direct method (without derivatization) was developed for the detection of reduced glutathione (GSH) in cultured hepatocytes (HepG2 cells) using high-performance liquid chromatography with electrochemical detection (HPLC-ECD). The method was validated according to the guidelines of the U.S. Food and Drug Administration in terms of linearity, lower limit of quantitation (LOQ), lower limit of detection (LOD), precision, accuracy, recovery, and stabilities of GSH standards and quality control samples. The total analysis time was 5 min, and the retention time of GSH was 1.78 min. Separation was carried out isocratically using 50 mM sodium phosphate (pH 3.0) as a mobile phase with a fused-core column. The detector response was linear between 0.01 and 80 μmol/L, and the regression coefficient (R(2)) was >0.99. The LOD for GSH was 15 fmol, and the intra- and interday recoveries ranged between 100.7 and 104.6%. This method also enabled the rapid detection (in 4 min) of other compounds involved in GSH metabolism such as uric acid, ascorbic acid, and glutathione disulfite. The optimized and validated HPLC-ECD method was successfully applied for the determination of GSH levels in HepG2 cells treated with buthionine sulfoximine (BSO), an inhibitor, and α-lipoic acid (α-LA), an inducer of GSH synthesis. As expected, the amount of GSH concentration-dependently decreased with BSO and increased with α-LA treatments in HepG2 cells. This method could also be useful for the quantitation of GSH, uric acid, ascorbic acid, and glutathione disulfide in other biological matrices such as tissue homogenates and blood.