The proximate analysis of the hoof of buffalo was carried out to assess the biochemical properties. The study revealed that buffalo hoof contained 14.78±0.38 percentage moisture. The crude protein (CP) content on dry matter basis was 88.04±0.08 percentage, total ash on dry matter basis was 1.84±0.03 percentage, crude fiber (CF) on dry matter basis was 0.79±0.01 percentage, ether extract (EE) on dry matter basis was 0.88±0.02 percentage and nitrogen free extract (NFE) on dry matter basis was 8.45±0.04 percentage. The findings about the biochemical composition of buffalo hooves will form a strong anatomical foundation on the structure of hoof of these animals, which can be used for extrapolations and further investigations in the same species or for comparative analysis, including cattle and horses. Another positive aspect was the possibility of associating the dry material, mineral matter, organic matter, crude protein and ester extract with the strength of the digits that bear the greater portion of the animal’s body weight and the impact when the hooves touch the ground.