Rationale: Diterpene lactones (DL) in Andrographis paniculata (AP) are known as "natural antibiotics" for their excellent antibacterial activity. During mass spectrometry (MS) analysis, the hydroxyl groups in the AP DL skeleton are prone to neutral loss of H 2 O, producing high in-source fragment peaks and affecting the characterization of these components.Methods: Mass tags were applied during the MS data acquisition step, and special adduct ion form was used to guide the data processing and characterization steps.Besides, the total number of characterized AP DLs significantly increased when combining the number of neutrally lost H 2 O from AP DLs, incorporating information on the diagnostic ions, and adopting molecular networks generated with the Global Natural Products Social Molecular Networking database.Results: Ninety-nine DLs, comprising 6 monohydroxyl groups, 20 dihydroxyl groups, 27 trihydroxy groups, and 46 DLs with more than 3 hydroxyl groups, were characterized from AP. In addition, based on the characteristic fragments in the product ions (C 3 H 4 , Δm/z = 40.03 Da), it could be assumed that 90 DLs had the C19-OH structure among the identified DLs. The current study provides a new approach for collecting, processing, and characterizing MS analysis of natural DLs prone to in-source fragmentation.Conclusions: MS characterization of AP DLs was significantly improved, and many potential new compounds were identified in AP. This characterization provides new methods for the purification and identification of AP DLs.