A new method for isolation and spectrophotometric determination of plumbagin is presented. Plumbagin was isolated by thin layer chromatography (TLC) and column chromatography (CC) techniques, as an orange tinged yellow long crystalline substances. Plumbagin exhibits two absrop-tion maxima at 410 and 510 nm. Stability of the color, pKa value, and the effect of pH were studied. Beer's law is obeyed over the range 0.9-45 ppm. The method is applied to the determination of plumbagin in roots, stems, and leaves of Plumbago europaea L. plant.