Pharmacognosy helps to authentify and standardise natural drugs. The present study attempts to characterise the bark of Vachellia nilotica (L.) P.J Hurter& Mabb. Subsp. indica (Benth.) Kyal. & Boatwr using microscopic characters, histochemical localisation, powder microscopic features, physico chemical parameters and HPTLC profiling. Bark showed the presence of cork, parenchymatous cortex, pericycle with lignified bands of stone cells, wide lignified phloem with prismatic calcium oxalate crystals and starch grains. Presence of tannin, starch and lignin were localised in the bark tissue. The HPTLC fingerprint of bark samples extracted in water, methanol + water (1:1), methanol, chloroform, acetone and petroleum ether using cold maceration technique and soxhlet method were obtained. Maceration method of extraction revealed maximum number of peaks in methanol+ water (1:1) extract while soxhlet method showed maximum peaks in acetone extract. Pharmacognostic parameters studied can be useful to lay down standards for the authentification of V. nilotica bark.