In this study, ultra performance liquid chromatography-quadrupole-time of flight tandem mass spectrometry (UPLC-Q-TOF-MS/MS), in combination with molecular networking technology based on the global natural products social molecular networking (GNPS) platform, was applied for the first time to systematically analyze and identify two classes of alkaloid bioactive components in the roots of Tripterygium wilfordii. This approach provides a solid foundation for the further development and application of T. wilfordii and its alkaloid-based bioactive constituents. The analysis was performed on a Hypersil GOLD™ C₁₈ column (100 mm × 2.1 mm, 1.9 μm), using a gradient elution with methanol-0.1% formic acid aqueous solution as the mobile phase, at a flow rate of 0.25 mL/min and a column temperature of 40 ℃. The mass spectrometric data of the 95% ethanol reflux extract of the roots of T. wilfordii were collected in positive ion mode, uploaded to the GNPS platform for computational analysis, and visualized molecular networks were constructed using Cytoscape 3.7.1 software. Based on reference standards, accurate molecular weights, characteristic fragment ions, and literature data, a total of 83 alkaloid components were successfully identified. Among them, 14 novel macrolide alkaloids, six novel macrocyclic polyamine alkaloids, and one novel 13-hydroxy macrocyclic polyamine alkaloid were identified. Notably, four macrolide alkaloids were identified for the first time from the Tripterygium genus. These results not only enrich the chemical profile of T. wilfordii but also provide clear guidance for the isolation, purification, and pharmacological evaluation of these novel compounds. Furthermore, the study validates the advantages and value of combining UPLC-Q-TOF-MS/MS with GNPS-based molecular networking for the efficient analysis and identification of complex natural products.