Purpose of the study: To develop a simple, rapid, and accurate HPLC method for simultaneous quant. determination of latanoprost, timolol, and benzalkonium chloride (BAK) in ophthalmic solution Chromatog. separation was achieved with PDA detector using Inertsil C18, 300 × 3.9 mm, 5μ reverse phase anal. column. The mobile phase consist of buffer:acetonitrile (40:60 volume/volume), was passed through the column at flow rate of 1.0 mL/min. The method was performed at wavelength gradient. The experiment was carried out at 30°C. The calibration curves were linear in the concentration range of 25% to 150% of the working concentration (r2 > 0.999). The lower limit of quantification was 0.8, 0.9 and 0.6 for timolol, BAK, and latanoprost, resp. The developed procedure was used for simultaneous quant. determination of latanoprost, timolol, and benzalkonium chloride (BAK) in ophthalmic solution Developed method was validated as per ICH Q2 (R1), and most useful for academic as well as industrial scale.