A reversed-phase liquid chromatography (RP-LC) method was validated for the determination of gatifloxacin in eye drops. The LC method was carried out on a Phenomenex LiChrosphere 5 µm RP-18 100 Ǻ 125 x 4.6 mm maintained at room temperature. The mobile phase consisted of water:acetonitrile:triethylamine (80:20:3 v/v/v), pH adjusted to 3.3 using phosphoric acid, run at a flow rate of 1 mL/min and using ultraviolet detection at 293 nm. The chromatographic separation was obtained with a retention time of 3.6 min and was linear in the range of 30-70 µg/mL (r² = 0.9991). The specificity and stability indicating the capability of the method was proven through forced degradation studies, which also showed that there was no interference of the excipients. The accuracy was 100.63% with RSD = 1.65. Method validation demonstrates satisfactory results for precision and robustness. The proposed method was applied for the analysis of marketed eye drops, for improving the quality control and to assure the therapeutic efficacy.