Abstract: A method was developed for the rapid and systematic identification of alkaloids in Chelidonium majus L. by HPLC-Q-TOF/MS. The separation was performed on an XCharge C18 column (5 μm, 4.6 mm×250 mm) with acetonitrile-0.1% formic acid in water by gradient elution. The flow rate was 0.7 mL·min^-1. The primary mass spectrometer molecular ion and the secondary mass spectrometry fragment ion were determined. The mass spectrometric cleavage of alkaloids was determined and the structures were used to systematically identify the alkaloids in Chelidonium majus L. To verify the accuracy of the qualitative results, three alkaloids were purified and identified by NMR. Twenty-one alkaloids were identified from Chelidonium majus L., including one aporphine-type alkaloid, three protopine-type alkaloids, 11 benzophenanthrine-type alkaloids and six protoberberine-type alkaloids. (S)-N-methylstylopine (8) was first reported in Chelidonium majus L. and dihydrocoptisine (11) and norchelidonine (12) were identified for the first time in Chelidonium majus L. using this technique. The chemical structures of the purified compounds are consistent with the qualitative results of the mass spectrometric analysis. The method is fast and accurate and can provide a basis for the identification and extraction of the chemical constituents of Chelidonium majus L.