The objective of the present study was to prepare, characterize and evaluation of gastroretentive microspheres of Cloxacillin. To achieve these objective nine formulations of microspheres were prepared by emulsion solvent evaporation method using Eudragit RS100 and Kollicoat MAE 100 P polymer. Prepared microspheres were evaluated for Particle size analysis, Surface morphology, Fourier transform infrared spectroscopy analysis, Drug encapsulation efficiency, Percent buoyancy, In-vitro drug release study and Stability study. The microspheres formed have smooth surface and spherical in shape as observed in scanning electron microscopy. The drug entrapment efficiency of the formulation is in the range of 76.46% to 93.41%. Particle size increases with increasing concentration of polymer, particle size ranging from 3 to 20 μm. The percent buoyancy was more than 70% up to 12 hours. The percent buoyancy was increased significantly with increase in polymer is in the range of 72.40% to 83.49%. The Drug-polymer compatibility was studied by using FTIR spectroscopy. The study revealed that there is no interaction between the drug and selected polymers. The drug release study was found to be controlled release manner and release kinetics follows peppa’s model and non-fickian in nature. The stability studies data was found that there was no such difference in drug entrapment efficiency and in-vitro drug release. So it indicates that biocompatible and cost effective polymers like Eudragit RS 100 and Kollicoat MAE 100 P polymer can be used to formulate efficient microspheres with good percentage entrapment efficiency and controlled release up to 24 hr in phosphate buffer pH 7.4. Hence these microspheres of Cloxacillin can be targeted to gastroretension.