Abstract: Due to the characteristics of small dosage, strong efficacy, fast metabolism in the body, and low content of novel designer benzodiazepines, it was difficult to detect them in the blood and urine of victims. Hair had the advantages of long detection time, easy acquisition, and easy preservation. Therefore, the title method was proposed. 20 mg of hair cut to 1-2 mm was taken, and 1 mL of extract containing internal standard (1 μg·L^-1 alprazolam-d₅) was added. The mixture was pre-cooled at -50℃ for 5 min, frozen and ground twice (each time for 90 s, with an interval of 60 s), and centrifuged for 5 min. The supernatant was taken and concentrated to dryness, and the residue was redissolved in 100 μL of methanol. The solution was filtered by 0.22 μm organic filter membrane filtration, and the filtrate was separated on the Acquity UPLC HSS T3 column, with a mixture of 0.1% (volume fraction, the same below) formic acid solution and acetonitrile solution containing 0.1% formic acid at different volume ratios as the mobile phase for gradient elution. The electrospray ion source positive ion (ESI⁺) mode was used for scanning and multiple reaction monitoring (MRM) mode was used for detection, with qualitative analysis by retention time and characteristic ions and quantitative analysis by internal standard method. As shown by the results, linear range of working curves for 7 novel designer benzodiazepines (remimazolam, clonitrazolam, flualprazolam, bromazolam, flubromazolam, etizolam, and brotizolam) was 1-500 pg·mg^-1, with detection limits (3S/N) in the range of 0.11-0.35 pg·mg^-1. Test for recovery was made by standard addition method, giving results in the range of 90.2%-104%, with RSDs (n=6) relative peak areas in the ranges of of 1.7%-4.7% for intra-day precision test and 2.9%-7.0% for inter-day precision test, respectively.