Gentamicin-Eudragit RS100 microspheres were prepared by modified double emulsion method. A 3(2) full factorial experiment was designed to study the effects of the composition of outer aqueous phase in terms of amount of glycerol (viscosity effect) and sodium chloride (osmotic pressure gradient effect) on the entrapment efficiency and % yield and microsphere size. The results of analysis of variance test for responses measured indicated that the test is significant (p>0.05). The contribution of sodium chloride concentration was found to be higher on entrapment efficiency and % yield, whereas glycerol produced significant effect on the mean diameter of microspheres. Microspheres demonstrated spherical particles in the size range of 33.24-60.43 microm. In vitro release profile of optimized formulation demonstrated sustained release for 24 h following Higuchi kinetics. Finally, drug bioactivity was found to remain intact after microencapsulation. Response surface graphs are presented to examine the effects of independent variables on the responses studied. Thus, by formulation design important parameters affecting formulation characteristics of gentamicin loaded Eudragit RS100 microspheres can be identified for controlled delivery with desirable characters in terms of maximum entrapment and yield.