This study investigates the role of buffalo colostrum and milk whey derived peptides in protection against oxidative damage induced by 2, 4-Dinitrophenol (2, 4-DNP) in human blood serum samples in vitro. A biomarker enzymes for oxidative stress like Alkaline phosphatase (ALP) and acid phosphatase (ACP), oxidative damage markers indicating extent of host antioxidant reserve indicators like reduced glutathione (GSH) were measured by spectrophotometric techniques in four different groups namely (1) Human blood plasma only (control),(2) Human blood plasma+ 2, 4-DNP,(3) Human blood plasma+ colostrum whey derived peptides+ 2, 4-DNP,(4) Human blood plasma+ colostrum whey derived peptides,(5) Human blood plasma+ milk whey derived peptides+ 2, 4-DNP and (6) Human blood plasma+ milk whey derived peptides. Following exposure to 2, 4-DNP, levels of antioxidants like GSH was significantly decreased in comparison to control, eg, GSH [0.568±0.015 vs0. 871±0.022 µmol/0.1 mg proteins. In addition, the concentration of biomarker enzymes for 2, 4-DNP induced membrane damage and oxidative stress like ALP and ACP were increased in serum by oxidant compared to control, eg, ALP [5.497±0.185 vs. 2.782±0.000 µmol/mg protein], ACP [1.689±0.047 vs. 0.629±0.047 µmol/mg protein]. Pretreatment with buffalo whey derived peptides significantly protects 2, 4-DNP induced RBC membrane lyses and release of ALP and ACP into serum environment. eg, ALP [3.444±0.094 vs. 5.497±0.185 µmol/mg protein], ACP [0.629±0.047 vs. 1.689±0.047 µmol/mg protein]. Pretreatment with whey derived peptides give protection to oxidative damage and shifts the trend towards amelioration and replenishment of the antioxidant status.