Background: gamma-hydroxybutyric acid (GHB), notorious as a club-and date-rape drug, was quantified in dried blood spots (DBS) by punching out a disc, followed by 'on-spot' derivatization and ana-lysis by GC-MS. Results: A homogenous distribution in DBS was demonstrated and accurate results were obtained when analyzing a disc punched out from a 20-35 mu l spot, regardless the hematocrit of the blood sample. Validation based on US FDA and European Medicines Agency guidelines was performed, with a calibration range covering 2-100 mu g/ml. Conclusion: A sensitive GC-MS method for GHB ana-lysis in DBS was successfully optimized and validated. The successful ana-lysis of DBS collected from GHB abusers suggests the routine applicability of the DBS sampling technique for GHB ana-lysis in toxicological cases.