A reversed-phase HPLC-DAD method for determination of menadione (MN) by converting menadione sodium bisulphite (MSB) to MN in rabbit plasma was developed and validated. C8 column with gradient elution of methanol and water with DAD detection at 330 nm in chromatographic separation and the one-step extraction method of MN from plasma matrix with n-hexane using menatetrone (MT) as internal standard (IS) was used. The calibration curve of MN was linear between concentration ranges of 0.061-12.2 μg/mL. Average recovery of MN and IS from the biological matrix was obtained approximately 96.3% and 98.9%, respectively. The intra-day and inter-day precision and accuracy were between 1.35-3.12% and 2.79-6.45%, respectively. In addition, the proposed method was successfully applied to the pharmacokinetic study by converting MSB to MN in New Zealand white rabbits receiving an intravenous high-dose injection of 100-mg MSB.