Abstract: Objective To establish an ultra-high performance liquid chromatography coupled with quadrupole-time of flight mass spectrometry（UPLC/Q-TOF-MS）method for rapid screening of toxic substances in Datura stramonium L. Methods After the samples were extracted by methanol, the liquid to be tested was separated on ACQUITY UPLC? BEH C18 column (2.1mm×50mm, 1.7μm) with 0.01% formic acid water as mobile phase A and acetonitrile as mobile phase B. The sample volume was 2.0μL, the column temperature was 35℃, and the flow rate was 0.2μL/min. ESI positive ion mode collection, mass spectrometry with precise molecular weight and chromatographic peak retention time for qualitative analysis, [M]+H as quantitative ion for quantitative analysis. Results The linear ranges were 1.0-200.0μg/L with r 2 ≥0. 990. The limits of detection were 0.08-2.24 ng/L the limits of quantification were 0.27-7.46 ng/mL. The recoveries were in the range of 78%~89% and RSDs were ≤3.0%.The contents of scopolamine 0.65mg/kg, anisodamine 0.66mg/kg and hyoscyamine 0.72mg/kg were detected in Datura stramonium L collected in the field. Scopolamine 0.0048mg/kg, anisodamine 0.041mg/kg and hyoscyamine 0.16mg/kg were detected in the liquor of Datura stramonium L. The target compounds were detected in different degrees in the blood and urine of the poisoned patients. Conclusion The method established in the laboratory is fast, efficient and accurate, which is suitable for the qualitative and quantitative detection of toxic substances in the poisoning incidents of Datura stramonium L. Two poisonings were caused by consumption of Datura stramonium L.