Rifampicin (RIF) is a semi synthetic derivative of rifamycin B. RIF is crystalline and hydrophobic in nature which may limit therapeutic concentration of RIF at the target site. Crystallanity of RIF can be reduced by formation of nanoparticles. Aim of the present investigation was to develop vanillin crosslinked chitosan nanoparticles of rifampicin by 32 factorial design. Effect of concentration of chitosan and crosslinker on Particle size (PS) and Entrapment efficiency (EE) was studied. PS of nanoparticles was determined by Dynamic Light Scattering method. EE was determined by centrifugation method and free drug was analyzed by UV-visible spectrophotometry. The optimised nanoparticles were characterised by Fourier Transform Infrared Spectroscopy (FTIR), Differential Scanning Calorimetry (DSC), X-Ray Powder Diffractometer (XRPD), scanning electron microscopy and in vitro release study. EE of RIF nanoparticles were found to be in the range of 48.76±0.95 % to 83.24±1.68%. The PSs of the nanoparticles were found to be in the range of 89.63±9.78nm to 436.4±8.44nm. Coefficients of regression equation suggest that PS and EE of chitosan nanoparticles are dependent on concentration of chitosan and vanillin. Optimum experimental conditions were selected based on the overlay plot of PS and EE. Value of average percent error was found to be sufficiently low which confirms the validity of model. FTIR, DSC and XRPD study revealed the formation of nanoparticles. SEM study indicates formation of porous nanoparticles. Results of the study indicate that vanillin can be used as a crosslinker for the preparation of RIF loaded chitosan chitosan nanoparticles. © RJPT All right reserved.