This project focuses on developing an efficient and sensitive method for detecting steroid esters in dried blood spots (DBS) to support anti-doping efforts. By optimizing a fully automated DBS processing system with an independent desorption pump, we established a high-throughput screening method for 30 steroid esters and a confirmatory method for 26 targets using HPLC-HRMS. The method demonstrates low detection limits (as low as 0.05 ng/mL) and good reproducibility (RSD < 15%), and its performance was validated through blind samples, administration studies, and comparison with traditional IRMS. This approach provides a practical solution for rapid and accurate screening and confirmation of steroid esters, enhancing anti-doping capabilities.