BACKGROUND: Milk-derived protein hydrolysates have generated a great deal of interest recently due to their numerous beneficial health effects. However, there are few comparative studies on protein hydrolysates from different dairy species, their production, characterization, and bioactivity. In the present study, skimmed milk from both major and minor dairy species was hydrolyzed with alcalase, and its protein profiles were studied using tricine polyacrylamide gel electrophoresis and reverse phase-high protein liquid chromatography. The antioxidant and in vitro immunostimulatory properties were determined. RESULTS: Iron chelation activity was highest in hydrolysates of whey (25.00 ± 0.32 mmol L −1 ), casein (25.14 ± 0.34 mmol L −1 ), colostrum (24.52 ± 0.28 mmol L −1 ), and skimmed cattle milk (24.21 ± 0.26 mmol L −1 ). ⊍,⊍-Diphenyl-⊎-picrylhydrazyl scavenging and 2,2 0 -azobis(2-amidino-propane) dihydrochloride activity was lowest in skimmed donkey milk protein hydrolysates (MPHs) (IC 50 : 5.37 ± 0.05 mg mL −1 and 151.59 ± 2.1 mg mL −1 ). Production of nitric oxide and phagocytosis activity in RAW 264.7 (murine macrophage cell line) was significantly higher among whey and buffalo skimmed milk protein hydrolysatetreated groups as compared with the untreated group. The incorporation of whey protein hydrolysate and skimmed buffalo milk protein hydrolysate were sensorially acceptable at 10% level in beverage mix.CONCLUSION: This study comparatively evaluates the antioxidative and immunomodulatory properties of different skimmed MPHs and their potential applications as ingredients in pediatric, geriatric, and other health-promoting foods.