When interpreting gamma-hydroxybutyric acid (GHB) concentrations in post-mortem specimens, a possible increase in GHB concentrations because of post-mortem generation must be considered. In this study, endogenous GHB concentrations in post-mortem biological fluids were investigated. Additionally, we review post-mortem GHB concentrations already published in the literature. Heart and peripheral blood samples, cerebrospinal fluid, urine, and vitreous humor were collected from 64 autopsies in subjects where the cause of death excluded GHB exposure. Sample analysis was carried out either on the day of autopsy or later after immediate freezing and storage at -20 °C. GHB concentrations in venous blood samples (n = 61) were <0.6-28.7 mg/L (mean 11.9 mg/L; median 10.6 mg/L), <0.6-65.3 mg/L (mean 15.2 mg/L; median 12.8 mg/L) in heart blood (n = 56), <0.6-25.1 mg/L (mean 6.0 mg/L; median 3.8 mg/L) in urine (n = 50), <0.6-39.0 mg/L (mean 9.6 mg/L; median 7.5 mg/L), in vitreous humor (n = 54), and <0.6-24.0 mg/L (mean 4.2 mg/L; median 3.2 mg/L) in cerebrospinal fluid (n = 52). There was no significant difference between GHB concentrations in cases where there were signs of beginning putrefaction at the time of autopsy (n = 9) and cases without obvious signs of putrefaction. In one case with advanced putrefaction, the GHB concentration in venous blood was 32.7 mg/L. In conclusion, for post-mortem venous blood, urine, and cerebrospinal fluid, an interpretative cut-off of 30 mg/L for GHB concentrations is suggested in cases where GHB analysis is conducted on the day of sample collection at autopsy or if samples have been stored at -20 °C immediately after collection.