This research presents the development and validation of a robust, precise, and accurate reverse-phase high-performance liquid chromatographic (RP-HPLC) method for the simultaneous determination of Erythromycin and Isotretinoin in tablet dosage forms. The chromatographic separation was achieved using an Inertsil ODS C18 column (4.6 × 250 mm) with a mobile phase composed of phosphate buffer (pH 4.6) and acetonitrile in a 30:70 (v/v) ratio, pumped at a flow rate of 1.0 mL/min. The column temperature was maintained at 30°C, and detection was performed at 260 nm with a run time of 7 minutes. The retention times for Erythromycin and Isotretinoin were found to be 2.395 min and 3.906 min, respectively. The method demonstrated excellent linearity in concentration ranges of 1-5 μg/mL for Erythromycin and 100-500 μg/mL for Isotretinoin, with correlation coefficients (r²) of 0.999 for both analytes. The percentage recovery was 100.1% for Erythromycin and 100.4% for Isotretinoin, with relative standard deviations (RSD) of 0.31% and 0.38% for repeatability, and 0.12% and 0.15% for intermediate precision, respectively. The limit of detection (LOD) values were 2.94 μg/mL and 3.03 μg/mL, while the limit of quantification (LOQ) values were 9.87 μg/mL and 10.1 μg/mL for Erythromycin and Isotretinoin, respectively. The method was fully validated according to ICH guidelines and found to be specific, accurate, precise, and robust, making it suitable for routine quality control analysis of pharmaceutical formulations containing these active pharmaceutical ingredients.