Animal urine contains different non-polar volatile compounds, which are known to stimulate their sexual behavior. These compounds collectively termed as pheromones that remain bound to some urinary proteins, which help in their signaling. The objective of this experiment was to identify the urinary volatile compounds before and after protease treatment in bull and in various reproductive stages of female buffaloes, viz. estrus, diestrus and pregnancy, by chemical extraction followed by thin layer chromatography (TLC). Buffalo bull urine showed different compounds in TLC upon protease treatment, where as no change in retention time values were observed for female buffalo urine upon protease treatment. It was found that buffalo bull urine contains protein bound non-polar compounds, which can be set free upon protease treatment and detected by TLC.