Background: Ciprofloxacin hydrochloride-encapsulated poly[LA-(Glc-Leu)] copolymeric nanoparticles were prepared to provide sustained release and ameliorate shortcomings associated with eye drops. Methods: Poly[LA-(Glc-Leu)] copolymer was synthesized by ring opening copolymerization of l-lactide with a cyclodepsipetide. Ciprofloxacin hydrochloride-loaded polymeric nanoparticles were prepared by double emulsification-solvent evaporation technique with emphasis on optimization of different process and formulation variables. Results: Optimized formulation showed encapsulation efficiency of approximately 63.08 ± 5.88% and a positive zeta potential of +17.46 mV. Scanning electron microscope study revealed the spherical shape of the nanoparticles with a particle size 300-400 nm. In vitro drug-release studies showed sustained release for 24 h with biphasic release pattern. In vivo ocular tolerability study in rabbits demonstrated no signs of toxicity or irritation parallel to the results obtained with the marketed formulation. Conclusion: Poly[LA-(Glc-Leu)] copolymer can be used as an efficient carrier for nanoparticle preparation of ciprofloxacin. © 2013 Future Science Ltd.