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A neuropsychiatric drug overdose impairs physiological function via central nervous system (CNS) depression. In drug-related deaths, only the drug concentration can currently provide information regarding CNS depression in victims. In this study, using a drug overdose model, we investigated the ability of neurotransmitters in the cerebrospinal fluid (CSF) to serve as biomarkers for CNS depression. Four groups of rats were orally administered diazepam (200 mg/kg) and/or phenobarbital (100 mg/kg) or vehicle. In a hot plate test performed to assess physiological impairment, drug-administered animals showed prolongation of the response latency. Serum drug concentrations were also sufficient to observe the effect of drug overdose. The levels of benzoyl-derivatized neurotransmitters were measured using liquid chromatography–tandem mass spectrometry (LC-MS/MS) analysis. Noradrenaline, adrenaline …