¹University Department of Chemical Technology, Dr. Babasaheb Ambedkar Marathwada University, Aurangabad, Maharashtra, India DOI : 10.29228/jrp.432 A simple, rapid and sensitive Reverse phase High performance liquid chromatography (RP-HPLC) bioanalytical method was developed for the determination of folic acid in rat plasma using methylparaben as an internal standard. The developed method involves simple sample preparations and had total run time of 20 min using RP-HPLC. Chromatographic separation was performed on Inertsil ODS C18 column (4.6×100 mm, 5μm) using isocratic elusion system of potassium phosphate buffer (pH 7): Methanol (75:25 v/v) as mobile phase. Mobile phase flow rate was kept at 1ml/min with column temperature of 40°Cat pressure 88-90 bars. The maximum wavelength used for the detection of folic acid was 283nm. The retention time of folic acid and methylparaben was found to be 6.4 & 13.2 respectively. The method was tested for linearity range of 2 to 12 μg/ml. Developed method was validated for system suitability, selectivity, linearity & range, accuracy, precision, robustness, LOD & LOQ, and stability according to the international regulatory guidelines. Minimum sample preparation and short run time make the method valuable for performing the analysis. Developed method was successfully applied in the determination of folic acid levels In-vivo in rat plasma after oral administration. Keywords : RP-HPLC, folic acid, methylparaben, validation, plasma