A rapid analytical method has been developed for the mutual resolution of three steroidal compounds, withaferin A, iochromolide, and withacnistin. Liquid chromatography was performed on a Chromolith analytical column (4.6 mm i.d.×50 mm), made from a cylindrical silica rod, operated at a flow rate of 4 mL min−1 with a simple linear gradient prepared from 0.1% aqueous formic acid and 0.1% formic acid in acetonitrile. Under optimum conditions simultaneous separation of the compounds was achieved in less than 7 min, one eighth the time required for conventional LC separation. The overall analysis time was reduced without sacrificing chromatographic performance—essential for the resolution of positional isomers such as iochromolide and withacnistin. The column was coupled to a single-quadrupole mass spectrometer and the method was characterized by good performance in terms of repeatability, selectivity, linearity, and sensitivity. Detection limits in the single-ion-monitoring mode were 0.15 μg mL−1 or below. Finally, the developed method was successfully applied to the determination of withanolides in extracts fromlochroma gesnerioides obtained by three different processes—traditional Soxhlet extraction and two faster methods, microwave-assisted extraction and pressurized solvent extraction.