High reproductive efficiency is a prerequisite for dairy animals in order to optimize dairy production. An accurate and early pregnancy diagnosis is a crucial aspect for better reproductive management in livestock. The indigenous, visual and clinical methods have various limitations including accuracy, sensitivity, specificity, later stages of applicability and requires highly skilled manpower. This hurdles the researchers to ensue further research on developing novel early pregnancy diagnostics for dairy animals. However, the advantage of molecular techniques like proteomics has given a new hope to look for pregnancy biomarkers in animal research. In this study, we assayed the pregnancy-associated glycoprotein (PAG) levels using anti-bPAG antibody. Serum plasma samples were collected 30, 60, 90 and 120 days after insemination. Cattle diagnosed pregnant with singleton are used for this experiment in which blood samples were collected. The plasma PAG level is gradually increased after insemination to until 60 days and drastically decreased after that. The PAG level was approximately 2-fold greater during 30 days compared with 60 days. These results concluded to validate our other pregnancy diagnosis methods including circulating microRNA and cell free DNA concentration (data not shown). Despite this proteomic approach, validating all other molecular technique results may give the exact time for early pregnancy diagnosis.