AbstractTraditional eye drops used for topically administering drugs have poor ocular bioavailability due to the biological barriers of the eye. There is an interest to design and develop novel drug delivery systems that would extend the precorneal residence time, reduce the frequency of administration and decrease dose-related toxicity. This study aimed to prepare Nanoparticles of Gemifloxacin Mesylate and incorporate them into anin situgel. The nanoparticles were prepared by ionic gelation technique, using 32factorial design. Sodium tripolyphosphate (STPP) was used to crosslink Chitosan. The optimized formulation of the nanoparticles (GF4) contained 0.15% Gemifloxacin Mesylate, 0.15% Chitosan and 0.20% STPP, producing 71 nm particle size and 81.11% entrapment efficiency. The prepared nanoparticles showed biphasic release, with an initial burst release of 15% in 1.0 hr and a cumulative drug release of 90.53% at the end of 24 hrs. After that, the prepared nanoparticles were incorporated into anin situgel, using Poloxamer 407, producing a sustained drug release with efficient antimicrobial activity against gram-positive and gram-negative bacteria as confirmed by the cup plate method.