Plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone) is an herbal ingredient which is isolated from the root of Plumbago zeylanica L. This herb is a semi-climbing subshrub distributed in thickets or grassland at low elevations of Taiwan. The crushed roots of P. zeylanica L. were ground from lumps to powder and boiled with H2O, 50% EtOH, or 95% EtOH. Chromatographic separation of plumbagin from the herb was carried out using a ZORBAX Extend-C18 column (150 x 4.6 mm I.D.; 5 microm) that was eluted with the mobile phase of water-methanol (10:90, v/v). Multiple reaction monitoring (MRM) was used to monitor the transition of the deprotonated molecule m/z 187 [M-H]- to the product ion m/z 159 [M-H-CO]- for plumbagin analysis. The limit of quantification was determined to and accuracy of 1 ng/ml. Furthermore, the mass fractions of plumbagin in P. zeylanica L. for H2O, 50% EtOH and 95% EtOH were 0.24 +/- 0.04, 3.92 +/- 0.87 and 13.4 +/- 1.59 g/kg, respectively. These results present a reliable liquid chromatography coupled with tandem mass spectrometric (LC-MS/MS) method for the determination of plumbagin form herbal medicines.