Saliva is considered as the best source for biomarker-discovery studies, since it is a non-invasive method when compared to other body sources. Usually, buffalo cannot express their estrus signs precisely. Hence, there is a need for concise methods to detect the time of estrus to ensure the success in artificial insemination. Therefore, we have established a reference proteome map on buffalo whole saliva during estrous cycle in order to document the estrus-specific proteins using SDS-PAGE and mass spectrometry. The present findings conclude that the proteomic approach adopted to identify the proteins from buffalo saliva around estrous cycle may provide a new tool for screening the estrus phase.