Abstract: A rapid determination of methamphetamine, amphetamine, 6-monoacetylmorphine, and morphine in hair samples by UPLC-MS/MS was established and optimized.The concentration of target compounds in the hair of drug abusers and drug laboratory technicians was investigated and the cut-off value was evaluated.After cleaned hair was extracted by grinding with methanol-water (7∶3) at 3 000 r/min for 100 s, the final solution after adjusting the volume to methanol-water (1∶1) was analyzed by UPLC-MS/MS.The analytes were gradient eluted on a Waters Acquity BEH C₁₈ (2.1 mm × 100 mm, 1.7 μm) column with 5 mmol/L ammonium formate-0.1% formic acid aqueous solution and acetonitrile as mobile phase at a flow rate of 0.4 mL/min. The ESI⁺ ion source and multiple reaction monitoring (MRM) were used to select the qualitative and quantitative ion pairs of the four target compounds. All analytes showed good linearity (R² ≥ 0.999 6) in the range of 0.01-5 ng/mg (except amphetamine in 0.01-4 ng/mg), limit of the quantitation was 0.01 ng/mg, and the limit of detection was 0.001-0.008 ng/mg.The accuracy, precision, matrix effect, and recovery all met the requirements of biological sample methodology.According to the comprehensive consideration of the receiver operating characteristic (ROC) curve, Youden index, law enforcement cost and intensity, the reference cut-off values were methamphetamine ≥ 0.1 ng/mg; amphetamine ≥ 0.025 ng/mg; 6-monoacetylmorphine ≥ 0.05 ng/mg; morphine ≥ 0.05 ng/mg.The method established in our research can quickly and accurately detect the contents of methamphetamine, amphetamine, 6-monoacetylmorphine, and morphine in hair.This study provides some reference for the public security system to make more rational cut-off values in the norm of drug-related personnel hair samples detection in the future.