AbstractThe possible performance‐enhancing effects and medical benefits of ecdysterone (ECDY) have been discussed several times throughout the last decades. In 2020, the World Anti‐Doping Agency include ECDY in their monitoring programme and continued this prevalence study until now. Only little is known about the human metabolism of ECDY besides the first study performed on human subjects in the field of sports drug testing that was already conducted in 2001. Aim of this study was the in‐depth investigation on human ECDY metabolism to improve its detectability and to support the decision‐making processes as to how ECDY can be implemented most effectively into sports drug testing regulations. In a first trial, one male volunteer was administered with threefold deuterated ECDY to enable the detection and potential identification of all urinary metabolites still comprising the deuterium label by employing hydrogen isotope ratio mass spectrometry and high‐resolution/high‐accuracy mass spectrometry. Samples were collected for up to 14 days, and metabolites excreted unconjugated, glucuronidated, and sulphated were investigated. The detected deuterated metabolites were confirmed in a second administration trial encompassing two male and one female volunteers. After the administration of 50 mg of unlabelled ECDY, urine samples were collected for up to 7 days. Besides the already described urinary metabolites of ECDY, more than 20 new metabolites were detected encompassing all expected metabolic conversions including side chain cleavage at C21. A large interindividual variation in the amounts of excreted metabolites was visible, and considerable differences in abundances of early‐ and late‐excretion phase metabolites were observed.