Abstract: Khat is a kind of common drug plant. Many experiments have shown that the content of cathinone norpseudoephedrine and norephedrine in some varieties of Khat are similar. However, some studies showed that there are differences in the intensity of psychoactivity between these varieties. Therefore, it is speculated that other substances in Khat may affect the psychoactive strength of Khat. But currently there are some difficulties in non-targeted screening of unknown compounds without mass spectrometry database information and standard solutions. In this paper, the unknown cathinone alkaloids in Khat were discovered and resolved by HPLC-Q-TOF MS in-source fragmentation multistage mass spectrometry. The separation was carried out on a ZORBAX Eclipse Plus Phenyl-Hexyl column (3.0 mm×100 mm×1.8 μm) with a gradient elution using 0.1% formic acid in water (A)-acetonitrile (B) as the mobile phase. The mobile phase flow rate was 0.3 mL/min, and the column temperature was 30 ℃. The screening of unknown cathinone alkaloid components was realized by the in-source cleavage characteristics of cathinone alkaloids in single-stage mass spectrometry under full-scan mode using an ESI ion source, and then the molecular structures of the unknown cathinone alkaloid components were deduced from the multi-stage MS cleavage patterns of the unknown cathinone alkaloid components in the target ion acquisition mode. The results showed that this method can effectively realize the screening and resolution of the unknown alkaloid components in Khat. A total of three cathinone alkaloid components, including methcathinone, ethcathinone and 2-amino-1-phenyl-1-butanone, were identified in Khat. Methcathinone and ethcathinone were corroborated using standard substances, and 2-amino-1-phenyl-1-butanone needs to be further corroborated due to the lack of corresponding standard substances. The work provides a guideline for the further study of the psychoactive components in Khat, and a reference for the analysis of unknown compounds in other fields.