Withania somnifera, popularly known as ÂAshwagandhaÂ, is one among the top rankingmedicinal plants of India and is highly valued for itsmedicinal and neutraceutical properties. It holds a position of importance similar to ginseng in China. The medicinal properties of Withania somnifera were attributed to the presence of steroidal lactones collectively termed as withanolides present in both the leaves and tuberous roots that can be used for treating various diseases. In the present study, an attempt has been made to analyze the variation of withanolide content in methanol extracts prepared from different parts of Withania somnifera namely root, rhizome head and stembyHPLCanalysis.HPLCchromatogramrevealed that similar peaks were found at the retention time of 7.7 and 9.9 minutes for all the samples. Concentration of withanolides calculated using the area count of similar peaks obtained at the retention time of 7.7 and 9.9 minutes revealed that Withania somnifera roots has more withanolides compared to the stem and rhizome head portions.