To establish a solid phase extraction (SPE)/high-resolution benchtop Q Exactive mass spectrometry (HRMS) method for rapid analysis of muscarine in human urine.Methods Samples were pre-concentrated and cleaned up directly with SPE. The chromatographic analysis was performed on an HSS T₃ column with 0.1% formic acid and 4 mmol/L ammonium formate in water/acetonitrile as the mobile phase with gradient elution, and external standard calibration was used for quantification. Results The linearity was within 0.05-5 μg/L, and the coefficient of determination (r) was not less than 0.999 for muscarine. The limit of detection (LOD) and the limit of quantitation (LOQ) were 0.02 and 0.05 μg/L, respectively. The recoveries of the spiked samples at the levels of 0.05,0.1, and 0.5 μg/L ranged from 73.2% to 97.9%, with the relative standard deviations (RSDs) from 2.5% to 10.2%.Conclusion The proposed method is rapid, simple, sensitive and specific enough for the rapid analysis of muscarine in human urine, which is sufficient to meet the requirements of clinical medicine and forensic toxicology.