A simple method for simultaneous determination of gatifloxacin and prednisolone acetate in ophthalmic formulation was developed and validated using UV spectrophotometry. Gatifloxacin and prednisolone acetate were quantified using the first-order derivative of the UV spectra. The proposed method was validated according to the guidelines of the International Conference on Harmonization and the Association of Official Analytical Chemists International. The measurements were made in acetonitrile/water (70:30 v/v) at 348nm for gatifloxacin and at 263nm for prednisolone acetate. The calibration curves were linear in the concentration range of 3–21μgmL−1 for gatifloxacin and 6–42μgmL−1 for prednisolone acetate with Sandell’s sensitivities of 0.349μgcm−2 and 0.402μgcm−2, respectively. The mean recovery and the limit of quantification for gatifloxacin were 99.76±0.41% and 1.11μgmL−1 and for prednisolone acetate were 99.52±0.87% and 0.55μgmL−1, respectively. The method was precise, with a relative standard deviation of less than 2.50% for both drugs. For robustness, the factors analyzed did not significantly affect the quantification of gatifloxacin and prednisolone acetate. The results of the validated method did not differ significantly from high-performance liquid chromatography (HPLC), which was previously developed and validated for the same drugs. In this form, the method was suitable for routine analysis of gatifloxacin and prednisolone acetate in their combined dosage form in ophthalmic formulations.