Aim: Quantification of testosterone (T) and 5α-dihydrotestosterone serum concentrations proved to be an efficient alternative to urinary steroid profiling for the detection of T doping. In this context, additional serum markers could be discovered by exploratory untargeted steroidomics studies. Results: Endogenous steroid metabolites were monitored by ultra high-performance liquid chromatography coupled to high-resolution mass spectrometry in serum samples collected during a T administration clinical trial. A three-step workflow for accurate review of annotation was used and multifactorial data analysis allowed highlighting promising serum biomarkers. Longitudinal monitoring of selected compounds was performed to assess T abuse detection capabilities. Conclusion: Application of serum steroidomics showed high potential for biomarker discovery of T doping, suggesting longitudinal monitoring of steroid hormones in serum as a significant improvement in detection of endogenous steroids abuse.