Gramicidin, Neomycin, and Triamcinolone are commonly used to treat bacterial infections in the eyes, rheumatoid arthritis, osteoarthritis, and skin disorders. The three components are determined simultaneously using ultra performance liquid chromatography. Waters Aquity ultra performance liquid chromatograph with photo diode array detector was used to develop and validate the method. The chromatographic separation was accomplished with a phenyl column (100 x 2.1mm, 1.7μm) and a mobile phase containing a 30:70 mixture of Methanol and Buffer (1.36g KH2PO4 dissolved in 1L distilled water). Before being used for analysis, the mobile phase is filtered through a 0.45μm filter. The auto sampler was kept at room temperature throughout the experiment. The injected sample volume is 5 μl, with a mobile phase flow rate of 0.5 l/min. The eluate was analyzed at 222nm, and the total runtime for analysis is 2.40 minutes. According to ICH guidelines, the research findings for system acceptability, reproducibility, detection limits, quantitation limits, linearity, accuracy, precision, and specificity are all within acceptable bounds.