Early pregnancy diagnosis is necessary to maintain reproductive efficiency in animals. To determine biomarkerfor the early pregnancy diagnosis, total protein was estimated and SDS PAGE was performed on urine and serum inselected 30 Sahiwal cows. Total serum protein concentration was increased in pregnant as compared to non-pregnant Sahiwal cows. A continuously increasing trend of protein was observed in pregnant group after 12 (6.36 ± 0.76 g/dl) to 22 (6.87± 0.39 g/dl) days post-breeding whereas non-specific trend was found in non-pregnant group. In urine, total protein concentration increased significantly in pregnant group as compared to non-pregnant group from 16 to 22 days. An increase in protein concentration from day 0 to 16 followed by decrease till day 22 was observed in both groups. Maximum and minimum protein concentration was observed on day 16 (31.6 ± 0.68 mg/dl) and day 0 (23.32 ± 0.81 mg/dl) respectively, in urine of pregnant group. Upon SDS PAGE analysis of serum, expression of specific proteins of molecular weight between 29 to 43 kDa with over expression of 66 kDa protein were observed in pregnant cows. It was observed that, proteins with molecular weight of 43 kDa and 66 kDa were over-expressed in pregnant cows as compared to non-pregnant cows. These findings suggest that pregnancy specific proteins ranging 29 to 43 kDa of pregnant Sahiwal cows can be used as pregnancy biomarker in near future