Tobramycin and Dexamethasone are widely used in combination ophthalmic formulations for the treatment of ocular infections and associated inflammation. Tobramycin is an aminoglycoside antibiotic with potent activity against gram-negative and some gram-positive bacteria, while Dexamethasone is a corticosteroid that suppresses inflammatory responses. This study presents a validated high-performance liquid chromatographic method for the estimation of Tobramycin and Dexamethasone in pharmaceutical ophthalmic solutions using a C18 reversed-phase column. The mobile phase for Dexamethasone consisted of methanol: water (75:25 v/v) at a flow rate of 0.7 mL/min. For Tobramycin, a Tris buffer system was used as the mobile phase at a flow rate of 1.0 mL/min. Due to Tobramycin’s weak UV absorbance, samples were derivatized using chemical methods to enhance its detection. Both drugs were effectively separated and derivatized under the optimized chromatographic conditions. The method was validated according to ICH guidelines, demonstrating good linearity, accuracy, precision, specificity, and robustness. Results were also compared to the official methods described in the USP, where individual estimations are performed and it is suitable for routine quality control of combined pharmaceutical formulation.