As the antioxidant and free radical scavenger, glutathione (GSH) participates in the preservation of cellular redox status and defense against reactive oxygen species and xenobiotics. Glutamate-cysteine ligase (GCL; also known as ${gamma}$-glutamylcysteine synthetase, EC 6.3.2.2) is the rate limiting enzyme in GSH synthesis. In the present study, the accurate method for determination of GCL activity in crude liver extracts was developed by measuring both ${gamma}$-glutamylcysteine and GSH from cysteine in the presence of glutamate, glycine and an ATP-generating system. We added glycine to promote the conversion of ${gamma}$-glutamylcysteine to GSH, and to minimize the possibility of ${gamma}$-glutamylcysteine metabolism to cysteine and oxoproline by ${gamma}$-glutamylcyclotransferase. We established optimal conditions and substrate concentrations for the enzyme assay, and verified that inhibition of GCL by GSH did not interfere with this assay. Therefore, this assay of hepatic GCL under optimal conditions could provide a more accurate measurement of this enzyme activity in the crude liver extracts.