A fast, sensitive and accurate reverse phase liquid chromatographic method was developed and validated for the simultaneous determination of Dorzolamide and Timolol maleate in ophthalmic preparations. Chromatographic separation was achieved on Inertsil ODS 3V C18 column (250 X 4.6 mm, 5 µm particle size) with mobile phase consisting of Acetonitrile and 1-Octane Sulphonic acid buffer (0.02M) pH adjusted to 3.5 ± 0.05 with o-phosphoric acid (36:64 V/V) at a flow rate of 1.0 mL/min. The analytes were detected at 254 nm and 295 nm for Dorzolamide and Timolol maleate respectively by PDA detector. Brimonidine was used as internal standard (IS). The retention time of Dorzolamide, Timolol maleate and Brimonidine were found to be at 6.020 ± 0.02, 8.254 ± 0.01 and 4.636 ± 0.01 mins respectively. The linearity of the method ranged between 4-720 and 1-180 µg/mL for Dorzolamide and Timolol maleate respectively with correlation coefficient 0.999 for both the drugs in binary mixture. The LOD was found to be 0.6951 µg/mL and 0.2489 µg/mL for Dorzolamide and Timolol maleate respectively and LOQ was found to be 2.3214 µg/mL and 0.8317 µg/mL for Dorzolamide and Timolol maleate respectively. Keywords: Dorzolamide, Timolol maleate, RP‐HPLC, Simultaneous estimation.