In this study, Remdesivir concentrations in rat plasma were accurately quantified using a validated and robust bioanalytical method based on Reverse Phase High Performance Liquid Chromatography (RP-HPLC). The method complied with stringent US FDA guidelines, demonstrating excellent accuracy, sensitivity, selectivity, and precision. Efficient sample recovery through simple protein precipitation enhanced the reliability and effectiveness of the procedure. Statistical analysis confirmed the method’s suitability for routine commercial analysis and bioequivalence studies involving Remdesivir in both bulk drug and formulated products. This validated method provides a reliable tool for pharmacokinetic investigations and quality control assessment of Remdesivir formulations, ensuring the drug’s therapeutic integrity and performance.