Ecdysteroids are of interest as potential sport performance enhancers, due to their anabolic effects. The current study aimed to analyze levels of the most abundant ecdysteroid, ecdysterone (20‐hydroxyecdysone, 20‐OHE) in easily available dietary supplements, and, outline an analytical strategy for its detection, and that, of its metabolites, (1) following administration of pure 20‐OHE to uPA(+/+)‐SCID mice with humanized liver, (2) in a human volunteer after ingestion of two supplements, one with a relatively low, and the other a high, concentration of 20‐OHE, and, (3) to estimate the prevalence of use of 20‐OHE in elite athletes (n = 1000). Of the 16 supplements tested, only five showed detectable levels of 20‐OHE, with concentrations ranging from undetectable up to 2.3 mg per capsule. Urine of uPA(+/+)‐SCID urine showed the presence of 20‐OHE and its metabolite, 14 deoxy ecdysterone, within 24 hours (hr) of ingestion. In humans, both the parent and the metabolite were detectable within 2 to 5 hr of ingestion, with the metabolite being detectable for longer than the parent. After ingestion of a low dose supplement, the parent and metabolite were detectable for 70 and 48 hr, while following the higher dose it was 96 and 48 hr, respectively. Analysis of urines from athletes (n = 1000) confirmed four positives for 20‐OHE, suggesting a prevalence of use of 0.4%. Prevalence of its use by elite athletes was relatively low, however, this needs to be confirmed in other populations, and with other related ecdysteroids. Ecdysteroids have recently been included in the WADA Monitoring program due to their anabolic effects. Concentrations of 20 OHE in dietary supplements are variable. Urinary 20‐OHE and 14‐deoxy‐20‐OHE were detected in uPA(+/+)−SCID mice within 24 hours (hr) of ingestion. In humans, 20‐OHE and its metabolite were detectable for 70 and 48 hr (2 caps of 0.0088 mg) and for 96 and 48 hr (two capsules of 2.3 mg). In elite athletes there was a prevalence of use of 0.4%.