In the present work, Neem leaf extracts from Maharashtra and Telangana were compared by using RP-HPLC for difference in chemical concentration. The chromatographic conditions were successfully developed for the separation of Maharashtra extract by using Kromosil RPC18 4.6×150mm 5µm, flow rate was 1.0ml/min, mobile phase ratio was ACE: methanol (75:35% v/v) pH-3 (pH was adjusted with Orthophosphoric acid), detection wavelength was 260 nm. The instrument used was WATERS HPLC Auto Sampler, Separation module 2690, photo diode array detector 996, Empower-software version-2. The retention times were found to be 2.223 min and 2.324 min. The chromatographic conditions were success fully developed for the separation of Telangana extract by using Agilent C 18(4.6×150mm) 5µ, flow rate was 1.0 ml/min, mobile phase ratio was Methanol: ACN (60:40% v/v) pH 3 (pH was adjusted with orthophosphoricacid), detection wavelength was 265nm. The instrument used was WATERS HPLC Auto Sampler, Separation module 2695, photo diode array detector 996, Empower-software version-2. The retention times were found to be 2.229 min and 3.330 min. The results of the anti microbial activity studies suggest that the samples from Maharashtra and Telangana have a broad spectrum of antimicrobial activity and this effect is increased by increasing the quantity of this compound, which can be used as an alternative for antibiotics. KEYWORDS: Antibacterial activity, Azadirachta Indica, RP-HPLC, etc.