AbstractEstra‐4,9‐diene‐3,17‐dione (dienedione) is an anabolic androgenic steroid (AAS) sold as a bodybuilding supplement. It is prohibited in both human and equine sports. With no report of 4,9‐diene configuration in endogenous steroids, dienedione has long been considered a synthetic AAS. Nevertheless, the reoccurring detection of dienedione in colt (entire male horse) urine samples lead to the investigation of its possible endogenous nature in horses. This paper describes (i) the detection of naturally occurring dienedione in colts, (ii) the conjugation study of dienedione and (iii) the population study of free and glucuronide‐conjugated dienedione in colt urine. Qualitative and quantitative analyses of dienedione content in colt urine were performed, employing liquid chromatography–tandem mass spectrometry (UPLC–MS/MS).Qualitative analyses showed that dienedione was endogenous in colt urine and mainly in the form of glucuronide conjugates. Glucuronidation of dienedione was believed to happen at 3‐enol leading to dienedione‐3‐glucuronide. Upon the population study of free and glucuronide‐conjugated dienedione in colt urine samples (n = 175), the mean ± SD was determined to be 2.5 ± 3.5 ng/ml. The population data fitted a normal distribution after a fifth root transformation with the exclusion of one outlier by Grubb's test. A possible in‐house threshold was proposed at 30 ng/ml of free and glucuronide‐conjugated dienedione in colt urine associated with a risk factor of 1 in 14,269 (with a degree of freedom of 173). This is the first report of endogenous dienedione in entire male horses and the approach for controlling its potential misuse by using a threshold is also presented.