Response surface methodology utilizing the central composite rotatable design version was made use of to optimize formulation of ansamycin-loaded polymeric nanoparticles. The central composite rotatable design including three-factored factorial designs with 3 levels was utilized in this research study. The drug encapsulation efficiency, particle size and zeta potential of the nanoparticles were examined relative to 3 independent variables consisting of polymer concentration (X1), surfactant concentration (X2) and also proportion of organic to aqueous phase volume (X3). The outcome revealed that the ideal formula can be gotten from this response surface methodology. The ideal solution for the nanoparticles was made up of polymer concentration (X1) of 5% w/v, surfactant concentration (X2) of 1% w/v and also proportion of aqueous to organic phase volume (X3) of 10:1 v/v. Ansamycin nanoparticles under the optimized conditions generated the encapsulation efficiency of 89%, mean particle size of 121 nm and zeta potential value of − 25 mV. SEM of the optimized polymeric nanoparticle showed spherical particles. The in vitro experiments verified that ansamycin in the polymeric nanoparticles released progressively over the duration of 36 h. This research study revealed that the response surface methodology central composite rotatable design can successfully be gotten the modelling of ansamycin polymeric nanoparticles.