•SUPRAS method with 1-hexanol and THF was applied to pretreatment process of human urine and blood specimen.•Benzodiazepines and zolpidem in human urine and blood were extracted by our SUPRAS method for the first time.•This method was applied to human actual urine and blood samples.•Simplicity, rapidity, low cost, high recovery rate and good reproducibility were confirmed.•This study is of use for both clinical and forensic cases in benzodiazepines and zolpidem abuse. A high-throughput and sensitive method using supramolecular solvent (SUPRASs) for detecting 9 benzodiazepines and zolpidem in human urine and blood by gas chromatography-tandem mass spectrometry (GC–MS/MS) was newly established and applied to authentic human urine and blood samples in this study. Urine and blood samples were subjected to liquid–liquid extractions with supramolecular solvent mixture which consists of tetrahydrofuran and 1-hexanol. The solvent layer was evaporated to dryness by stream of nitrogen. The residue was reconstituted with methanol, and subjected to analysis by GC–MS/MS in multiple reaction monitoring (MRM) mode; internal standard method was employed for quantifying of each targeted compound. The regression equation has a good linear relationship with correlation coefficients for all tested compounds were not lower than 0.9991. The lower limits of the quantification ranged from 0.20 to 5 ng/mL for tested compounds in urine; Meanwhile, the lower limits of the quantification in this method ranged from 1 to 50 ng/mL for tested compounds in blood. These results showed that excellent reproducibility and satisfactory extraction recovery rates could be obtained for the established analytical method for 10 drugs in both blood and urine samples. The established method in this study was high-throughput, simple and sufficiently sensitive for determining of benzodiazepinesand zolpidem in human urine and blood. Therefore, this newly established method could be of use for qualitative and quantitative determination of such drugs in urine and blood samples either for clinical poisoning monitoring or for forensic identification.