Benzodiazepines (BDZ) and z-drugs belong to the most frequently prescribed medicines worldwide used for anxiety, epilepsy and sleeping disorders. Due to their pharmacology, they have a high potential for misuse. Hair analysis is being performed for the retrospective monitoring of drug exposure. However, there is a lack of reference values to obtain indication of BDZ/z-drug misuse. Further, there is no consensus on BDZ/z-drug cut-off concentrations above which a hair sample is reported as positive. The objective of the present study was to retrospectively evaluate BDZ/z-drug levels in hair for better interpretation of hair testing results. For this purpose, 4,630 authentic samples (head/body hair) from a heterogeneous cohort in Switzerland tested for the presence of 20 BDZ/BDZ metabolites and three z-drugs by liquid chromatography-tandem mass spectrometry were included. Drug concentrations in hair were statistically evaluated by box-plots in 1,726 positive samples. Further, metabolite-to-parent drug ratios were determined. Zolpidem, diazepam, nordazepam, oxazepam, lorazepam, midazolam, alprazolam, and bromazepam were among the most frequently detected drugs. Generally, drug concentration ranges varied strongly between the lower limit of quantification and 30,000 pg/mg hair. Sixteen BDZ/BDZ metabolites and zopiclone (z-drug) displayed a median below 50 pg/mg which is recommended as cut-off or minimum requirement for the limit of detection (LOD) by institutions. In case of ten drugs even the 75 percentile was below 50 pg/mg. Therefore, we strongly recommend to reconsider whether the use of an equal BDZ/z-drug cut-off is reasonable and whether minimum requirements for LODs should be lowered. The statistical evaluation of BDZ/Z-drug hair concentrations by box-plots can help in the development of analytical methods for hair samples and in the interpretation of BDZ/z-drug hair levels.Copyright © 2020. Published by Elsevier B.V.