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Background: The efficiency of reproduction in buffaloes can be increased by being able to spot buffaloes in estrus, as silent heat is a severe problem in this species. Proteins of urinary exosomes are identified to be better physiological biomarkers when compared to cell free proteins as these extracellular vesicles (EVs) are tiny in size, have additional protection from degradation by endogenous protease activity and are involved in intercellular communication. Hence, the present study was aimed to quantify the urinary exosome sex hormone binding globulin (SHBG) levels during estrus and diestrus stages of buffaloes to determine its suitability as a marker for identifying buffaloes in estrus. Methods: The grouping of animals as mid-diestrus (Group-I/G-I/Control group), regular estrus (Group-II/G-II) and silent estrus (Group-III/G-III) buffaloes was done using a combinatorial approach. The levels of SHBG in urinary exosomes of three groups of animals was quantified using the bovine SHBG ELISA kit. Result: The present study's findings showed significantly higher urinary exosome SHBG concentration (p<0.05) in G-I in comparison to G-II and G-III. The present study revealing for decreased concentration of urinary exosome SHBG in G-II and G-III animals when compared to G-I animals probably construes the physiological role of SHBG in the female reproductive tract at the estrus stage by mediating the action of estradiol 17 β (E2) at the target site. However, for urinary exosome SHBG to be considered as a biomarker, there is need for additional research on a bigger population. [ABSTRACT FROM AUTHOR]