Abstract Estrus detection in water buffalo is affected by the high incidence of poor manifestation inherent in this animal species. This affected the success rate of Artificial Insemination (AI) and Embryo Transfer (ET) due to the failure of perfect timing in AI and embryo-recipient synchrony in ET. While various ways of estrus detection were reported, the accuracy was interfered with by other symptoms that signify the health condition rather than the reproductive status of the animal. This results in a low conception rate and long calving intervals in AI and ET programs. Vaginal Cytology is a simple clinical tool of estrus detection that can also detect the pathological status of the reproductive tract and the perfect time for AI. While this is greatly used in canines, application in large animals was limited. The need to collect the swab from animals in the field and bring the smeared sample for analysis to the laboratory hindered its use and application. Our study, however, showed that by adopting this tool in estrus detection, increase in conception rate and calf production were achieved. A >60.0% cornification with the presence of >10 mm Graafian follicle is enough indicators of the perfect time for AI resulting in 80-100% conception. The development of a water buffalo vaginal chart helped determine the time of estrus. Strategies to adopt this tool for improved livestock reproduction are warranted.