Background:: Withanolide glycosides in Ashwagandha (Withania somnifera), are important metabolites attributed with widely acclaimed therapeutic potential for which validated methods for quantitative determination are limited. Objective:: The primary objective was to develop and validate a Reversed-Phase High Performance Liquid Chromatography (RP-HPLC) method for simultaneous quantification of total withanolide glycosides (WG), withanoside IV and withaferin A present in ashwagandha extract.The study also aimed to identify various other constituents present in the extract. Method: : Aqueous methanol extract (AME) of ashwagandha was prepared and fractionated into two viz. flavonoid rich fraction (FF) and withanolide rich fraction (WF). RP-HPLC method was developed and validated for estimation of total WG in ashwagandha extract according to ICH guidelines. Preparative HPLC based purification of major compounds from WF fraction was carried out and constituents were identified using spectroscopic techniques. HPLC chemical profiling of WF before and after acid hydrolysis under controlled conditions were carried out to further confirm the glycosidic compounds. Results: : The RP-HPLC method gave a precise differentiation of flavonoids, withanolides and WG present in ashwagandha extract. The method demonstrated good reliability and sensitivity, and can be conveniently used for the quantification of total WG, withanoside IV and withaferin A present in ashwagandha extracts. According to this method, purified fraction (WF) prepared from roots and leaves of ashwagandha comprise 35 % of total WG, 3.27 % of withanoside IV and 2.40 % of Withaferin A. The method was also applied to different products prepared from ashwagandha with total withanolide glycosides ranged from 1.5 % to 60 % and the results were found to be reproducible. Identification of the individual chemical constituents as well as the acid hydrolytic pattern of the extract further supported the reliability of developed method for the quantitative determination of total WG. This study also reported a new withanolide glycoside named, cilistol V-6’-O-glucoside (Aswanoside) along with some other known withanolide glycosides. Conclusion:: A Reversed-Phase High Performance Liquid Chromatography (RP-HPLC) method was developed and validated for quantitative determination of total WG, withanoside IV and withaferin A present in ashwagandha extract according to ICH guidelines. This study also reported a new withanolide glycoside named, cilistol V-6’-O-glucoside (Aswanoside) along with some other known WG.