A novel stability-indicating gradient reversed-phase ultra-performance liquid chromatographic (RP-UPLC) method was developed for the determination of purity of dorzolamide hydrochloride and timalol maleate in presence of their impurities, and forced degradation products and placebo. The method was developed using a Waters UPLC BEH C18, 100 × 2.1mm, 1.7 µm column with mobile phase containing a gradient mixture of solvents A and B. Phosphate buffer (0.04M), pH 2.6 was used as buffer. Buffer pH 2.6 was used as solvent A and Milli-Q water, methanol and acetonitrile in 200:300:600, v/v/v ratios were used as solvent B. The gradient program was set as 0/5, 8/8, 10/15, 16/45, 20/55, 24/80, 25/5 and 30/5. The eluted compound dorzolamide hydrochloride and its impurities were monitored at 254 nm, and timalol maleate and its impurities were monitored at 295 nm. The run time was 30 min, within which dorzolamide hydrochloride and its five impurities as well as timalol maleate and its three impurities were well separated, with resolution more than 2.0. Dorzolamide hydrochloride and timalol maleate were subjected to the stress conditions of oxidative, acid, base, photolytic and thermal degradation. The peak purity of dorzolamide hydrochloride, timalol maleate and their related compounds did not show any flag, thus proved the stability-indicating power of the method. The developed method was validated as per International Conference of Harmonization guidelines with respect to specificity, linearity, limit of detection, limit of quantification, accuracy, precision and robustness.