Recently, steroid hormones quantification in blood showed a promising ability to detect testosteronedoping and interesting complementarities with the urinary module of the Athlete Biological Passport(ABP). In this work, an ultra-high pressure liquid chromatography–high-resolution mass spectrometry(UHPLC-HRMS) method was developed for the quantification of eleven endogenous steroids in serum.The performance of the full scan and targeted SIM acquisition modes was evaluated and compared to theperformance of tandem mass spectrometry (MS/MS). Passing-Bablok regressions and Bland-Altman plotswere assessed for each analyte of interest, and concentration values measured by HRMS showed highcorrelation with the ones obtained by MS/MS for all target hormones, with low absolute differences in themajority of cases. A slight decrease in terms of sensitivity was observed with HRMS in both acquisitionmodes, but performing an analysis of variance multiblock orthogonal partial least squares (AMOPLS) onthe dataset obtained with all three methods revealed that only 0.8% of the total variance was related toinstrumentation and acquisition methods. Moreover, the evaluation of the testosterone administrationeffect over time highlighted testosterone itself and dihydrotestosterone as the most promising biomark-ers of exogenous testosterone administration. This conclusion suggests that HRMS could provide suitableperformance for blood steroid analysis in the anti-doping field.