Gamma-hydroxybutyrate (GHB) is a central nervous system depressant endogenously produced and also employed as a recreational drug of abuse since the 90s, attracting notable attention particularly in those cases of drug facilitated sexual assault (DFSA). The implication of GHB among DFSA cases generates numerous concerns, making the interpretation of the toxicological findings often significantly difficult. In this review the Authors have explored GHB behaviour in ante-mortem and post-mortem specimens, taking into consideration its endogenous production, the post mortem interval, the time between sampling and analysis, the storage conditions (temperature and presence/absence of preservatives) and the usefulness of alternative matrices such as hair, bile and vitreous humour. Moreover, the cut-off values currently recommended in forensic toxicology in order to discriminate between endogenous and exogenous levels have been examined.