A gas chromatographic-tandem mass spectrometric (GC-MS-MS) method for determining trace concentrations of gamma-hydroxybutyrate (GHB) in blood and urine has been developed. Multiple reaction monitoring was used to detect parent and daughter ions of GHB, 233 and 147, respectively, following liquid-liquid extraction with acetonitrile and derivatisation with N,O-bis[trimethylsilyl] trifluoroacetamide (BSTFA). Deuterated GHB was used as an internal standard. The assay produced excellent linearity and sensitivity without conversion to gamma butyrolactone. The lower limit of quantitation (LLOQ) in 50 μL of sample was 2.5 μg/mL. The expanded uncertainty values for intra- and interassay results were ± 0.097 and ± 0.123 ng/mL at a confidence level of 95% for blood and urine, respectively. Endogenous concentrations of GHB were found to be in the range of 0.3 to 6 μg/mL in urine and 0.5 to 2.3 μg/mL in blood, confirming previously suggested cut-off values for forensic analysis.