Gamma-hydroxybutyric acid (GHB) can be used as a knock-out drug in drug facilitated crime (DFC). Due to its rapid metabolism and resulting narrow detection window, uncovering GHB use in DFC still constitutes a problem. In this experiment we determined the GHB and GHB-β-O-glucuronide (GHB-Gluc) concentrations in hair samples after single and chronic GHB exposures. Hair samples of three narcoleptic patients therapeutically taking sodium oxybate (GHB-sodium-salt) were collected. Patients 1 (P1) and 2 (P2) took the medication for nine and six years, respectively. P1 took daily the pharmaceutical Xyrem(®) in a total dose of 5.78g GHB at bed time (2.89g) and four hours (2.89g) later. P2 took a dose of 3.10g GHB at bed time and an additional dose of 2.68g GHB four hours later. Patient 3 (P3) was newly diagnosed with narcolepsy and started his therapy with oral dose of 6g (divided in three portions of 2g GHB) within 24h. The hair samples were extracted both with and without forerunning washing steps. GHB and GHB-Gluc were determined by a published ultra-high performance liquid chromatography-tandem mass spectrometry method using GHB-d6 and GHB-Gluc-d4 as internal standards. GHB and GHB-Gluc concentrations in unwashed hair samples of P1 and P2 were determined in a range of 0.56-1.30ng/mg and <0.48-0.85ng/mg, respectively. In washed hair samples of P1 and P2 the concentrations were in a range of <0.32-0.68ng/mg and <0.48-1.20ng/mg for GHB and GHB-Gluc, respectively. The determined concentrations were within the published endogenous range. The confirmed results showed that the washing procedure before extraction causes a minor decrease of GHB concentrations in hair (difference: <1ng/mg). The investigations showed that a single GHB exposure might not be determined by hair analysis of GHB and GHB-Gluc. The chronical intake of therapeutic sodium oxybate with doses up to 7g per night was also not confirmed by hair analysis maybe due to hair treatments. Therefore, GHB hair analysis should be assessed critically and determined negative results could not exclude GHB exposures.