The quantification of the main withanolide, withaferin-A, in plant extracts of Withania somnifera and Withania coagulans using high-performance liquid chromatography (HPLC)-mass spectrometry (ESI-MS/MS) is described. HPLC was used in combination with a triple quadrupole mass spectrometer in multiple reaction monitoring (MRM) mode. Additionally, mass exact determination of withaferin-A was achieved using a hybrid high-resolution mass spectrometer (Q-TOF). A combination of acetonitrile and water as the mobile phase using gradient elution was found to be successful in separating the compound of interest from the other main components. The limit of detection (LOD) in the plants was 6 ng/g and the total analysis time was 10 min.