AbstractDried blood spots (DBSs) sampling is gaining support by the antidoping community because of simplicity and cost‐effective characteristics, especially in collection, transport, and storage. Nevertheless, DBS applicability demands specific studies for each of the analytes proposed for testing. Here, GHRP‐2 has been selected as a representing member of the growth hormone‐releasing peptides (GHRPs) family to provide further evidence of DBS suitability for GHRPs abuse detection in sport testing. An analytical procedure to extract GHRP‐2 and its main metabolite (AA‐3) from DBS and to detect them by liquid chromatography–tandem mass spectrometry (LC–MS/MS) has been developed. The method has been validated for the detection of GHRP‐2. Specificity and identification capabilities have been assessed in agreement with antidoping guidelines. The low AA‐3 levels found in DBS samples prevented its effective application for the determination of this metabolite. The limit of detection (LoD) for GHRP‐2 has been established at 50 pg/ml. Long‐term stability (>2 years) has been confirmed. The procedure has been successfully applied to actual DBS samples from an administration study with a single intravenous dose of GHRP‐2 (100 μg) being detected up to 4 h after drug injection. GHRP‐2 concentrations have been higher in venous blood DBS than in capillary blood DBS. Despite the observed differences, a similar detection window has been achieved independently of the type of blood used.In summary, this study provides specific evidence supporting DBS usefulness to detect GHRP‐2, and potentially other GHRPs family members, for antidoping tests.