The primary purpose of this study was to extend the time of interaction of the carrier with the negatively charged ocular surface for enhanced penetration in order to improve ocular bioavailability. Moxifloxacin hydrochloride (MOX) was successfully incorporated into cationic Eudragit (R) RS 100 nanoparticles (NPs) by spray-drying process. Particle size and zeta potential measurements, entrapment efficiency, morphological, thermal, FTIR and NMR analyses and MOX quantification by high performance liquid chromatography (HPLC) method were performed for characterizing the formulations prepared. In vitro release profiles indicated prolonged release of MOX from NPs which followed Korsmeyer-Peppas kinetic model. Cytotoxicity results showed that pure MOX and MOX-loaded NPs were not toxic at all concentrations for 24 h. Moreover, NPs remained stable for 3 months of storage under accelerated conditions. Conclusively, depending on the in vitro results obtained in this study, Eudragit (R) RS 100 NPs seem to be promising for enhancement of ocular bioavailability of MOX.