Updated project metadata. Background: An early, reliable and noninvasive method of pregnancy diagnosis is a prerequisite for efficient reproductive management in dairy industry. The early detection of pregnancy also helps to reduce the calving interval and rebreeding time which is beneficial for farmers and dairy industries. The aim of this work to identify potential biomarker for pregnancy detection at earlier stages (16-25 days). To achieve this goal, we performed differential in gel electrophoresis (DIGE) and label free quantitation (LFQ) for identification of protein which have significant differential expression during pregnancy. Results: DIGE experiment revealed eleven differentially expressed proteins out of which nine proteins were up regulated having fold change ≥1.5. The LFQ data analysis gave 202 differentially expressed protein out of 30 proteins were up-regulated and 40 down regulated having significant fold change ≥1.5 and ≤0.6 respectively. Further bioinformatic analysis showed that majority of proteins was involved in regulation of leukocyte immunity, endopeptidase inhibitor activity, regulation of peptidase activity and polysaccharide binding. Conclusion: To the best of our knowledge, this is first report on identification of differentially expressed proteins in urine of cows during various time points of pregnancy using DIGE and LFQ. In our investigation, we have discussed functional significance of few selected proteins such as A2HS, MBP, GRP, IGFBP-II, SERPIN, Vitamin D binding protein etc which were differentially expressed and actively involved in pregnancy associated events such as embryo implantation, establishment and maintenance of pregnancy. Thus, we have identified a set of potential protein biomarkers for early detection of pregnancy.