Abstract: Xylazine is used for sedation, anesthesia, and muscle relaxant as a veterinary drug and is not approved by the FDA for human use. Its chemical structure closely resembles the phenothiazine. But intoxication cases in human were identified due to its anesthetic effect in recent years, especially occurred in DFSA (drug-facilitated sexual assault) cases. So far, limited data concerning xylazine human metabolism have been reported. Urine is the preferred material for metabolite identification in forensic toxicology analysis because it can extend the detection time limit. In the last few years, high-resolution mass spectrometry (HRMS) such as time-of-flight (TOF) and Orbitrap MS have been widely used to discover and identify drug metabolites due to its high sensitivity, rapidity and mass accuracy. Drug metabolites can be rapidly detected and identified based on high resolution mass and multiple stage mass by high performed chromatography coupled with high-resolution mass spectrometry (HPLC-HRMS). To study the metabolites of xylazine in humans, the metabolism of xylazine was investigated in human urine after several sample workup procedures. For liquid-liquid extraction, the supernatant after mixing 1 mL of urine with 2 mL of ethyl acetate was first transferred, and further extraction was carried out by adding 1 drop of 10% NaOH and 2 mL of ethyl acetate. Then the two extracts were mixed, evaporated and reconstituted in 100 μL of methanol. For protein precipitation,100 μL of urine was precipitated by 900 μL acetonitrile. After shaking and centrifugation, 5 μL supernatant was taken for analysis. For this study, samples were separated on a Hypersil GOLD PFP column (100 mm×2.1 mm×3 μm), the mobile phase consisted of solvents A (5 mmol/L ammonium acetate solution with 0.1% formic acid) and B (acetonitrile). The analysis of metabolites were performed by liquid chromatography quadrupole-Orbitrap mass spectrometer (LC-Q-Orbitrap MS) with positive electrospray ionization (HESI-Ⅱ) interface operation in the full scan-ddMS² ion mode. The metabolites were identified by the comparison of theoretical accurate molecular mass, isotopic pattern abundance and product ion characteristics obtained through Compound Discover software. By analyzing the fragment ion given in the MS² spectrum, 7 phaseⅠmetabolites were detected in liquid-liquid extraction, including hydroxylation, oxidation and S-oxidation, 8 metabolites were detected in protein precipitation, including 4 phaseⅡmetabolites, excreted as glucuronides and sulfates. As a result, a total of 13 phase Ⅰ and Ⅱ metabolites were detected in human urine. It is preliminarily clarified the metabolic pathways and major metabolites of xylazine in humans. This paper shows great value for clinical and forensic toxicology research.