Gamma-hydroxybutyric acid (GHB) is a drug of abuse, that interplays with a GABAergic system, resulting in an euphoric state and increased mood and impulses. Two cases of fatal mixed intoxications including GHB intake are presented here. In both cases, GHB was used together with multiple other drugs. Interpretation of GHB cut-off values are complicated in post-mortem analysis, because GHB can be post-mortem formed. The post-mortem GHB formation is dependent of the post-mortem interval (PMI) and the storage conditions of the samples. The GHB concentrations in urine are more stable compared to blood samples, when the samples are stored at the correct way at − 20 °C. Therefore, urine is the recommended matrix to analyze in toxicological screenings, since it allows more specific determination of exposure to exogenous GHB. Different cut-off values are used for matrices from living and deceased people. A cut-off value of 30 mg/L is recommended to discriminate between endogenous concentrations and concentrations resulting from exogenous GHB exposure. Moreover, post-mortem GHB formation can take place before sampling. However, when the samples are immediately stored at cooled conditions, no in vitro formation of GHB will take place. Urinary screening of GHB may serve as an initial screening for estimation of exposure of GHB in the body. However, additional quantitative GHB analysis in blood is required to estimate GHB exposure at the time of death. Furthermore, to obtain more reliable results for the ante-mortem GHB exposure, it may be useful to measure other biomarkers, like some GHB metabolites, especially in blood.