Abstract Benzodiazepines are widely used in the treatment of sleep and anxiety disorders, as well as epileptic seizures and alcohol withdrawal because of their broad therapeutic index and low cost. Due to their central nervous system depressant effects they are also often implicated in traffic accidents and drug-related intoxications. With an increasing number of designer benzodiazepines used in a recreational setting, there is a need for analytical methods to be able to quantify both the prescribed and designer benzodiazepines. A liquid chromatography–triple quadrupole mass spectrometry method was developed for the quantification of 34 prescribed and 20 designer benzodiazepines in plasma. Different sample preparation strategies, including protein precipitation, liquid–liquid extraction, solid-phase extraction and mini-QuEChERS, were tested. The best recoveries for all compounds of interest were obtained with a liquid–liquid extraction using methyl-tertiary-butyl-ether and 500 μL plasma. The method was fully validated according to the European Medicines Agency guidelines for all compounds, except pivoxazepam, which is included for qualitative purposes only. In-sample stability issues were observed for cloxazolam, both at ambient temperature and during long-term storage at −20°C. Due to the large number of compounds included, the simple and time-efficient sample preparation and the relatively inexpensive instrumentation used, the presented method can be readily implemented in both therapeutic drug monitoring and forensic analyses.