Dorzolamide Hydrochloride is used to lower the increased intraocular pressure in open-angle glaucoma and ocular hypertension. Timolol maleate is indicated in the treatment of elevated intraocular pressure in patients with ocular hypertension or open-angle glaucoma. In this article, a high performance liquid chromatography (HPLC) method with UV-Visible detector wavelength (254 nm & 295 nm) for Dorzolamide Hydrochloride & Timolol maleate was developed and validated. The mobile phase consisted of methanol : buffer (60:40), the pH was adjusted to 3 with glacial acetic acid, was run through a Column C-18, (EC 150/4.6 NUCLEOSIL 100-5), reverse phase analytical column. The experiment was carried out at room temperature for Dorzolamide Hydrochloride & Timolol maleate. Analytical run time was less than 10 min. The assay exhibited good linear relationship. Accuracy & Precision were over the concentration range of 10 to 30 mg/ml & 40 to 120 mg/ml for Dorzolamide Hydrochloride and Timolol Maleate respectively. This method was found to be applicable for determination of the Dorzolamide Hydrochloride & Timolol maleate in active pharmaceutical ingredient (API) and pharmaceutical product also.