Background: Azacitidine is a demethylating agents and an antineoplastic agent used to treat myelodysplastic syndrome. Method: The RP-HPLC method for analysis of Azacitidine was developed and validated as per ICH guidelines. The separation of Azacitidine was performed on Inertsil C18 column (4.6×250mm, 5µ) with PDA detection carried out at 265nm. A box-behnken design with response surface methodology was executed out for optimization of chromatographic conditions of RP-HPLC for finished desired chromatographic estimation of Azacitidine with less number of experimental trials. Three independent factors namely 1%urea composition in the mobile phase, pH of an aqueous phase and flow rate were used to construct a mathematical model and study the effects of these independent factors on responses such as retention time, theoretical plates, tailing factors Results: Optimized experimental conditions for proposed work consists of 1%urea at pH 9.15 as mobile phase, at flow rate of 1ml/min with retention time was found to be 1.899. Then accuracy study was completed at three different levels and was found in the range 100 to 102%. The percent RSD value for precision was found to be 0.116. LOD and LOQ value for Azacitidine was found to be 0.0013 & 0.00416µg/ml. Conclusion: The 3D response surface graphs revealed that 1%urea composition and pH of aqueous phase were both most stringent factors affecting the responses. A rapid novel precise and accurate RP- HPLC method was developed and validated and can be used for regular analysis for the estimation of Azacitidine.