In this work, an RP-HPLC method was established for the simultaneous quantification of antazoline sulfate and naphazoline nitrate in commercial ophthalmic formulations. The proposed method was validated for sensitivity, selectivity, linearity, accuracy, precision, and stability, and was found to be suitable for routine analysis of these two active ingredients in the presence of degradation products. The optimized conditions using a C18 column, a mobile phase (phosphate buffer: methanol, 80:20), a flow rate of 1.5 mL/min, and detection at 285 nm at room temperature. The quantification method for antazoline sulfate and naphazoline nitrate in the pharmaceutical formulation was validated in accordance with International Conference on Harmonization (ICH) Q2 (R1) guidelines for the quantitative measurement of these pharmaceuticals. Under optimal conditions, a linear relationship with high correlation coefficients (0.9999 for Antazoline Sulfate and 0.9997 for Naphazoline Nitrate) was established between the concentration ranges of 0.11 to 0.35 mg/mL for Antazoline sulfate and 0.006-0.02 mg/mL for Naphazoline nitrate. The detection limits of Antazoline sulfate and Naphazoline nitrate were determined to be 0.3 and 0.06 mu g/mL, respectively. The developed method can be useful for quality control and stability testing of ophthalmic formulations containing antazoline nitrate and naphazoline sulfate.