The present study aimed to design, develop, and optimize an analytical procedure to perform the quantitative determination of ecdysterone in commercially available dietary supplements. The newly developed procedure is based on the extraction of ecdysterone from the supplements and the subsequent analysis by an optimized UHPLC-MS/MS method. Chromatographic separation was performed on an Agilent Eclipse Plus C18 column (2.1 mm x 100 mm, particle size 1.8 μm). The mass spectrometer was operated in positive ionization mode (ESI+) with acquisition in dynamic multiple reaction monitoring (dMRM) mode. Using the protonated molecular ion [M+H]+ ecdysterone (target) and cortisol (internal reference) were detected at m/z 481 and 363, respectively. The assay was fully validated according to ICH guidelines and the method resulted to be fit for purpose in terms of accuracy and precision (CV% and RE% <15). Time-different intermediate precision was found within the reported range according to AOAC guideline for dietary supplements and botanicals. Quantitation has been performed using an external calibration considering the minimal matrix influences, preliminarily assessed following a cross comparison with an elaborate and time consuming standard addition method. The method was successfully applied to 12 different dietary supplements labelled to contain ecdysterone, showing an actual content generally much lower than the labelled one. Copyright © 2019 Elsevier B.V. All rights reserved.