Objective. The aim of the study was to develop and validate a reversed-phase high-performance liquid chromatography (HPLC) method for the separation and quantification of two active pharmaceutical ingredients isohydrafural (IHF) and methyluracil (MU) in ear drops. Methods. An efficient separation of the examined compounds was achieved at 30 °C on a Teknokroma C1 5μm (150*4,6mm), with methanol and water 60:40 (V:V) and flow of 0.6 ml/min. The total test time was 5.5 min. Result. The tested validation parameters were: specificity, linearity, selectivity, precision (repeatability and reproducibility), robustness, limit of detection and limit of quantification. Conclusions. The results of the validation procedure (high recoveries, good standard deviations, no interference peaks at the retention times corresponding to the analytes) confirm that the developed chromatographic method can be applied for the routine analysis of combined ear drops with isohydrafural and methyluracil.