The research work was driven to develop novel nanostructured liquid crystalline particles of vancomycin for its improved pre-ocular residence time, ocular bio-availability, enhanced targeting, increased permeability, reduced dosing frequency, controlled drug release and reduced systemic side-effects.Formulation was developed by fragmenting cubic crystalline phase of glycerol monooleate, water and poloxamer 407. A four-factor, three-level Taguchi statistical experimental design was constructed to optimize the formulation. Formulations exhibited internal-cubic structure of the vesicles with particle size in the range of 51.11 ± 0.96 nm to 158.73 ± 0.46 nm and negative zeta potential. Ex-vivo transcorneal permeation studies demonstrated that the optimized cubosomes had 2.4-fold increase in apparent permeability co-e cient as compared to vancomycin solution. Whereas, in-vivo studies in rabbits demonstrated that the severity of keratitis was considerably lowered in day 3 with optimized cubosomes.Ocular pharmacokinetic studies evaluated level of drug in aqueous humor and results revealed that the time to peak concentration (T max ) of vancomycin loaded cubosomal formulation was about 1.9-fold higher and mean residence time was 2.2-fold greater than vancomycin solution. Furthermore, histological examination revealed that the corneal layers displayed well-maintained morphology without any stromal swelling, consequently indicating safety of formulation. In conclusion, results manifested that the developed vancomycin loaded cubosomes could be a promising novel ocular carrier and an ideal substitute for conventional eye-drops for the management of bacterial-keratitis.