Structural elucidation and gas-phase fragmentation of ten withanolides (steroidal lactones) were studied using a positive ion electrospray ionization quadropole time-of-flight mass spectrometry (ESI-QqTOF-MS/MS) hybrid instrument. Withanolides form an important class of plant secondary metabolites, known to possess a variety of biological activities. Withanolides which possess hydroxyl groups at C-4, C-5, C-17, C-20, and C-27, and an epoxy group at C-5/C-6, were evaluated to determine the characteristic fragments and their possible pathways. ESI-QqTOF-MS (positive ion mode) showed the presence of the protonated molecules [M + H]+. Low-energy collision-induced dissociation tandem mass spectrometric (CID-MS/MS) analysis of the protonated molecule [M + H]+ indicated multiple losses of water and the removal of the C-17-substituted lactone moiety affording the [M + H–Lac]+ product ion as the predominant pathways. However, withanolides containing a hydroxyl group at C-24 of the lactone moiety showed a different fragmentation pathway, which include the loss of steroidal part as a neutral molecule, with highly diagnostic ions at m/z 95 and 67 being generated from the cleavage of lactone moiety. Our results also determined the influence of the presence and positions of hydroxyl and epoxy groups on product ion formation and stability. Moreover, the knowledge of the fragmentation pattern was utilized in rapid identification of withanolides by the LC/MS/MS analysis of a Withania somnifera extract. Copyright © 2010 John Wiley & Sons, Ltd.