In anti-doping analysis, detecting long-term metabolites is essential foridentifying the misuse of banned substances. My project focuses ondeveloping and optimizing an LC-QQQ-MS method to determine THMT-sulfate, asulfoconjugated phase II metabolite of methyltestosterone, in biological samples. Methyltestosterone is an anabolic androgenic steroid that isprohibited both in and out of competition by WADA, yet it remains one ofthe most commonly used substances in competitive sports. Using a Design of Experiments (DoE) approach, I systematically assessedand refined key method parameters to enhance sensitivity, selectivity, and robustness, ensuring a reliable analytical approach for doping analysis. After optimization, the method will be validated and applied in acontrolled human excretion study to further evaluate its performance. My research aims to improve doping control strategies and advance analytical techniques in sports drug testing.