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According to WADA guidelines, the presence of Higenamine (HG) in urine should not be ≥10 ng/mL. HG is widely found in materials used in Chinese herbal medicines as well as food and additives. This paper is the first method wherein a rat model has been used to evaluate the pharmacokinetics of orally administered HG by LC-MS/MS and would be helpful in doping control analysis. The method was found to be linear over a concentration range of 0.5(lower limit of quantification, LLOQ)-500 ng/mL for plasma and 0.5(LLOQ)-1000 ng/mL for urine. The values for intra- and inter-day accuracy and precision did not deviate by >12.25% for HG in plasma and 5.87% in urine. Extraction recoveries of HG were 70.30-86.71% from plasma and 74.93%-79.29% from urine. HG was stable in plasma and urine after the extraction process and when exposed to different storage conditions. The findings of this study could provide some reference value for the assessment of HG misuse and for the control of intake and external application of HG-related materials (foods and medicinal herbs). Our key findings are that high levels of external application or oral administration of HG-rich materials may lead to a positive urine test for HG in athletes.