This study was designed to compare the effects of diets prepared with enzymatic hydrolysate of a whey protein concentrate (WPC) by pancreatin, protamex (Novo Nordisk, Bagsvaerd, Denmark), and alcalase proteases on the hepatic glutathione content in mice. The undenatured WPC was produced in a pilot plant by membrane technology (microfiltration/diafiltration) after separation of the casein clot through a conventional process. All three hydrolysates with 20% degree of hydrolysis showed an amino acid profile similar to WPC. Male A/J mice were fed on diets containing 20% WPC or hydrolysates. Commercial casein was used as a reference protein in the biological assays. The glutathione content was determined after liver extraction through high-performance capillary electrophoresis. WPC and its pancreatin and protamex hydrolysates showed higher ability to stimulate liver glutathione synthesis than alcalase hydrolysate. This difference was probably related to an amino acid sequence in the peptides that were formed during hydrolysis of whey proteins. Commercial casein and WPC alcalase hydrolysate produced lower stimulation of liver glutathione synthesis (7.09 and 5.66 micromol/g of wet weight) compared with WPC and pancreatin and protamex hydrolysates (8.72, 8.71, and 8.45 micromol/g of wet weight, respectively). These results indicate that the hydrolysates obtained by treatment with pancreatin and protamex are good sources of peptides with activity to stimulate glutathione synthesis.