Ecdysterone is a phytosteroid widely discussed for its various pharmacological, growth-promoting, and anabolic effects, mediated by the activation of estrogen receptor beta (ERbeta).Performance-enhancement in sports was demonstrated recently, and ecdysterone was consequentlyincluded in the Monitoring Program, to detect potential patterns of misuse in sport. Only fewstudies on the pharmacokinetics of ecdysterone in humans have been reported so far. In thisstudy, post-administration urine samples in twelve volunteers (single dose of 50 mg of ecdys-terone) were analyzed using dilute-and-inject liquid-chromatography–tandem mass spectrome-try. Identification and quantitation of ecdysterone and of two metabolites, 14-deoxy-ecdysteroneand 14-deoxy-poststerone, was achieved. Ecdysterone was the most abundant analyte present inpost-administration urine samples, detected for more than 2 days, with a maximum concentra-tion (Cmax) in the 2.8–8.5 h urine (Cmax= 4.4–30.0μg/mL). The metabolites 14-deoxy-ecdysteroneand 14-deoxy-poststerone were detected later, reaching the maximum concentrations at 8.5–39.5 h(Cmax= 0.1–6.0μg/mL) and 23.3–41.3 h (Cmax= 0.1–1.5μg/mL), respectively. Sex-specific differ-ences were not observed. Cumulative urinary excretion yielded average values of 18%, 2.3%, and1.5% for ecdysterone,14-deoxy-ecdysterone, and 14-deoxy-poststerone, respectively. Ecdysteroneand 14-deoxy-ecdysterone were excreted following first-order kinetics with half-lives calculated withthree hours, while pharmacokinetics of 14-deoxy-poststerone needs further evaluation.