Withanolides are important secondary metabolite in Withania somnifera (L.), which have high medicinal values and possess potent anti-tumor and antioxidant properties. A simple, rapid and specific reversed-phase HPLC method for the simultaneous analysis of nine structurally similar withanolides, namely, Withanolides A, B, IV, V, withanone, 27-hydroxywithanone, withaferin A, withastramonolide and physagulin D of root tissues of Withania somnifera, collected from Egypt, has been developed using a linear binary gradient solvent system comprising of (A) 0.14 g of potassium dihydrogen phosphate in 900 mL of water, 0.5 mL of phosphoric acid, dilute with water to 1000 mL, and (B) acetonitrile were used to profile the extract compositions and to quantify the withanolides therein. Homogeneity and purity of each peak was ascertained by comparative evaluation of the on-line UV spectra of the eluted compounds with those of the reference compounds. The method described can be easily and reliably applied to the quantitative analyses of withanolides in roots of Withania somnifera L.