Dried blood spots (DBS) provide a valuable complementary sample matrix for routine doping analysis, and the full automation of analysis for DBS samples is achievable to avoid extensive and repetitive manual laboratory work. In the current study, a fully automated online DBS preparation and detection method for the screening and quantification analysis of 13 anabolic steroid esters by means of the DBSA-TLX-HRMS system was developed and validated, based on the purpose for the determination of endogenous anabolic steroids administered exogenously. Validation of the method yielded linear (R2 > 0.99), precise and accurate (RSD% and Re% < 20% at low, medium, high concentration levels) results. The LOD of testosterone laurate was established at 0.5 ng/mL, and at 0.2 ng/mL for other steroid esters. The extraction recovery of the target compounds from DBS ranged from 10.5% to 88.9%, and matrix effects were moderate. Furthermore, the developed and validated method was applied in the analysis of DBS samples collected after the oral administration of a single dose of 80mg testosterone undecanoate demonstrating its applicability. Evaluation of analyte stability showed that testosterone undecanoate are more stable (8 weeks) in DBS samples of administration study when stored in frozen (-20°C) condition compared to cold storage (4°C). Collectively, these findings demonstrate the applicability of DBS sampling in doping control for detection of anabolic steroid esters.