According to the World Anti-Doping Agency (WADA) Prohibited List, glucocorticosteroids are prohibited in competition and only when administered by oral, intravenous, intramuscular or rectal routes. Up to now, in order to differentiate whether glucocorticosteroids were administered by one of the prohibited routes or not, a specific reporting limit for urinary concentrations of parent compounds and their metabolites was established at 30 ng/mL. Additionally, the new specific regulation starting from 1 September 2014 for budesonide have been introduced that the 6β-hydroxybudesonide shall be targeted. Budesonide is a glucocorticosteroid used mainly by inhalation for asthma management. Interestingly, anti-doping laboratory statistics show that budesonide adverse analytical findings (AAF) constitute almost 50% of all reported glucocorticosteroid AAFs, even though budesonide possesses a very low systemic activity which may cause performance enhance effects. This work presents the results of five studies of controlled budesonide administration carried out on professional athletes. The samples were analyzed by using a quantitative HPLC/MS/MS method for 16α-hydroxy-prednisolone, the most abundant budesonide metabolite in urine. Our data clearly show that inhalation of budesonide at least 12 h before a competition at therapeutic doses leads to appearance of the main budesonide metabolite in concentrations exceeding prior reporting limit for this compound. Therefore, our work strongly supports recent WADA decision not to target the main budesonide metabolite using the same reporting limit as for other glucocorticosteroids.