ABSTRACT The bioavailability of drugs from conventional eye drops is generally low. Many studies have demonstrated that new and more complex ophthalmic drug forms exhibit advantage over traditional ones and are able to increase the bioavailability of the active substance by, among others, reducing the susceptibility of drug forms to defense mechanisms of the human eye, extending contact time of drug with the cornea, increasing the penetration through the complex anatomical structure of the eye, and providing controlled release of drugs into the eye tissues, which allows reducing the drug application frequency. In this study, lipid-based microsuspensions of gentamicin were developed and investigated as alternative for ophthalmic delivery of gentamicin. Lipid matrices used were prepared by fusion using 1:1, 1:2 and 2:1 mixtures of Phospholipon ® 90G and Softisan ® 154. Gentamicin (0.1, 0.3, 0.5 and 0.7 w/w %) was incorporated into the lipid matrices and microsuspensions were formulated by melt homogenization technique. The microsuspensions for topical ophthalmic delivery were characterized in terms of particle size and morphology, thermal analysis, osmolarity, entrapment efficiency and loading capacity. The in vitro release study of gentamicin in phosphate buffer (pH 7.4) was carried out using polycarbonate dialysis membrane (MWCO 6000-8000) while the ex vivo permeation studies were conducted using excised pig cornea. The permeability coefficient and flux of the formulation across the excised cornea were determined. xvii The particle size of the formulations ranged from 9.15 ± 1.04 to 12.91 ± 0.5 μm. The microsuspensions had entrapment efficiency range of 25 - 64%, which were dependent on the concentration of drug. The osmolarity of the formulation was within the range of 280.33 ± 3.05 - 321.67 ± 2.08 mOsmol. The formulations were stable within the period of study. The lipid based formulations exhibited 49 - 88% drug release in vitro at 12 h and the release was dependent on the ratio of the lipids used. There was sustained permeability of the formulations through the excised cornea when compared with commercial gentamicin eye drop. The lipid based microsuspensions could be used for ophthalmic delivery of gentamicin.