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The main objective of this study was the development and optimization of fluorometholone-loaded PLGA nanoparticles for the treatment of inflammatory conditions of the eye. Design of experiments was used to obtain nanoparticles with the best physicochemical characteristics. The optimized nanoparticles containing 1.5 mg·mL−1 of fluorometholone showed a negative surface charge (−30 mV) and an average size below 200 nm being suitable for ocular administration. Drug-polymer interaction studies confirmed no new bonds were formed during the synthesis. Nanoparticles performance was assessed with biopharmaceutical behavior studies, ocular tolerance, anti-inflammatory efficacy and bioavailability. The biopharmaceutical behavior of the drug from nanoparticles was adjusted to hyperbola order showing a significantly greater permeation in the cornea than in the sclera. The optimized formulation had significantly greater anti-inflammatory effects than the commercial formulation. In addition, nanoparticles increased drug penetration toward the vitreous. Polymeric nanoparticles of fluorometholone could provide a suitable alternative for the treatment of inflammatory disorders of the anterior and posterior segments of the eye against of conventional topical formulations.