Buffalo skim milk retentate was hydrolyzed with papain for 4 h (enzyme:substrate, 1:200), resulting in a retentate hydrolysate (RH) with a degree of hydrolysis of 23%. We then investigated the potential hepatoprotective activity of RH at 250 and 500 mg/kg of body weight per day on carbon tetrachloride (CCl4)-induced oxidative stress in albino rats. Liver biomarkers (alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, and lactate dehydrogenase), kidney biomarkers (urea, creatinine), and serum lipid profile (total lipids and triglycerides) were measured, in addition to histopathological status. Injection of CCl4 significantly increased all liver and kidney biomarkers compared with the negative control. In contrast, CCl4 injection significantly reduced hepatic antioxidant enzyme activities; that is, glutathione peroxidase and superoxide dismutase. Oral administration of RH for 28 d effectively maintained a physiologically normal range of liver and kidney biomarkers compared with the positive control. Furthermore, RH administration significantly increased activities of glutathione peroxidase and superoxide dismutase. Histopathological sections of CCl4- stressed rats treated with RH were different from that of the positive control and were similar to those of the negative control, in a concentration-dependent manner. Our results demonstrated the antihepatotoxic activities of buffalo milk RH and demonstrated that the higher RH concentration (500 mg/kg of body weight per day) could maintain the healthy biological status of the CCl4-injected rats