Rationale : The in-source fragmentation (ISF) of analyte or co-eluting substances produces unintentional fragment ions, which hampers identification and quantification by liquid chromatog.-mass spectrometry (LC/MS). Natural compounds derived from plants also contain fragile moieties that may undergo ISF. However, the characteristics of ISF of natural compounds in LC/MS are still unclear. Methods : The ISF behavior of 214 natural compounds was assayed in LC with Q/orbitrap MS in electrospray ionization (ESI) mode and the extent of ISF was evaluated. Results : Up to 82% of tested compounds underwent ISF and half of the tested natural compounds that contain more than one fragile moiety underwent successive and severe ISF to generate serial structurally related ISF products. The major ISF-altering moieties for natural compounds were hydroxyl, lactone, glycosyl and ether, resulting in neutral loss of H2O or CO, deglycosylation or cleavage of ether bond, resp. Some compounds such as terpenoids underwent severe ISF and less than 1% parent form can be observed For natural compounds, ISF products with similar structures are more likely to cause interference in anal. because the ISF products may share identical mass-to-charge ratio and similar MS2 fragmentation patterns with precursor ions of the homologs in plants. Furthermore, severe ISF may cause a false neg. in the identification of the parent form. Conclusions : In summary, ISF was a highly frequent phenomenon for anal. of natural compounds by LC/ESI-MS, and extensive and successive ISF of natural products may cause misannotation and misidentification with homologs in plants. The study should raise awareness of ISF interference during the anal. of natural compounds