Background <p>The pharmacokinetic profiles of methylprednisolone (MP) in plasma and urine were evaluated after single and multiple oral doses. MP is prohibited in sports competitions when administered systemically (oral, injectable, or rectal administrations) whereas other routes remain permitted for therapeutic purposes.</p> Objective <p>The aim of this study was to characterize the urinary MP excretion after oral administration to assess whether the current World Anti-Doping Agency minimum reporting level of 30 ng/mL in urine is appropriate for distinguishing permitted from prohibited administrations, as well as whether the recommended washout period is adequate. Another aim was to better define MP elimination kinetics and evaluate its systemic effects.</p> Methods <p>Sixteen healthy male participants were enrolled and divided into two groups. One group received a single oral dose of 12&#xa0;mg of MP, while the other received 12&#xa0;mg daily for 3 consecutive days. Urine and blood samples were collected before, during, and after administration. Urine samples were analyzed for MP and 15 metabolites using a sensitive and selective liquid chromatography-tandem mass spectrometry method. Plasma samples were analyzed for unchanged MP and cortisol using liquid chromatography-tandem mass spectrometry. A pharmacokinetic analysis was performed using a one-compartment model with first-order absorption, combining plasma concentrations and urinary excretion data.</p> Results <p>MP represented only a small proportion of the administered dose, confirming that metabolism is the main elimination pathway. After a single oral dose, urinary MP concentrations above 30&#xa0;ng/mL were observed up to 12&#xa0;h, while all samples collected later were below this concentration. After multiple dosing, most samples above 30&#xa0;ng/mL were also found within 12&#xa0;h, although a few participants still showed concentrations above the minimum reporting level in the 12 to 24&#xa0;h period. All samples collected more than 24&#xa0;h after the last dose were below 30&#xa0;ng/mL. In plasma, MP reached peak concentrations around 1.6–2.3&#xa0;h after administration and was not detectable 24&#xa0;h after the last dose. Cortisol concentrations were markedly suppressed after dosing, with recovery to baseline within 24–48&#xa0;h depending on the regimen.</p> Conclusions <p>This study confirms that the current urinary minimum reporting level of 30&#xa0;ng/mL is appropriate for detecting oral MP use while minimizing the risk of false adverse analytical findings. The results also support the adequacy of the current 3-day washout period. Modern liquid chromatography-tandem mass spectrometry analysis and combined plasma-urine pharmacokinetic modeling provided robust evidence that MP is rapidly eliminated, extensively metabolized, and associated with transient cortisol suppression after oral administration.</p> Clinical Trial Registration <p>EudraCT number 2020-004596-41.</p>