Two simple, accurate, specific, and precise chromatographic methods, reversed phase high-performance liquid chromatography (RP-HPLC) and highperformance thin-layer chromatography (HPTLC), have been developed and validated for the determination of moxifloxacin hydrochloride and difluprednate in ophthalmic dosage form according to International Conference on Harmonization (ICH) guidelines. The separation of moxifloxacin hydrochloride and difluprednate in HPLC was performed on reverse phase (C18, 5 μm, 250 × 4.6 mm) column using isocratic condition, with acetonitrile, 5 mM disodium hydrogen phosphate buffer adjusted to pH 5, and methanol (50:25:25, v/v/v) as mobile phase. The flow rate for analysis was 1.0 mL min−1, and the selected chromatographic conditions effectively separated moxifloxacin hydrochloride and difluprednate with retention time of 3.6 and 6.6 min, respectively, at a detection wavelength of 254 nm. Chromatographic development in HPTLC was performed on precoated silica gel 60F254 aluminium plates with n-hexane, 6 M ammonia, and acetone (5:1.8:2, v/v/v) as mobile phase. The detection wavelength for simultaneous estimation of both drugs was 232 nm in HPTLC, and the Rf values for moxifloxacin hydrochloride and difluprednate were 2.2 and 7.1, respectively. The linear concentration range for HPLC method was 5 to 50 μg Ml-1 and 1 to 10 μg Ml-1; and for HPTLC method was 1200 to 2200 ng band-1 and 200 to 1200 ng band-1 for moxifloxacin hydrochloride and difluprednate, respectively. Moreover, Bartlett's test applied on the calibration peak areas revealed homoscedasticity of variance for both the methods. Both methods were validated with respect to system suitability, specificity, linearity, precision, accuracy, and robustness. The mean percentage recoveries for marketed formulation in terms of accuracy were found to be 100.53 and 100.58 for HPLC; and 100.56 and 100.30 for HPTLC for moxifloxacin hydrochloride and difluprednate, respectively. The pooled percent relative standard deviation (% RSD) value for repeatability, intermediate precision, accuracy, and robustness studies for both the methods were found to be less than 2. Result of paired t-test at 95% confidence level reveals that there is no significant difference between recoveries of drugs, using both methods. The results of the developed chromatographic methods were acceptable assuring that these methods can be successfully applied for routine quality control testing of both bulk and ophthalmic dosage forms, without any interference from the excipients.