Abstract: Codonopsis pilosula (CP), a well-known food medicine homology plant, is commonly used in many countries. In our preliminary study, a series of pyrrolidine alkaloids with high MS responses were detected as characteristic absorbed constituents in rat plasma after oral administration of CP extract. However, their structures were unclear due to the presence of various isomers and the lack of reference standards. In the present study, an MS-guided targeted isolation of pyrrolidine alkaloids of CP extract was performed by ultra-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (UPLC/Q-TOF MS). For data analysis under fast data directed acquisition mode (Fast-DDA), an effective approach named characteristic fragmentation-assisted mass spectral networking was successfully applied to discover new pyrrolidine alkaloids with high MS response in CP extract. As a result, seven new pyrrolizidine alkaloids codonopyrrolidiums C–I ( 3–9 ), together with two known ones ( 1 and 2 ), were isolated and identified by NMR spectral analysis. Among them, codonopyrrolidium B ( 1 ), codonopyrrolidium D ( 4 ) and codonopyrrolidium E ( 5 ) were evaluated for lipid-lowering activity, and they could improve high fructose-induced lipid accumulation in HepG2 cells. In addition, the characteristic MS/MS fragmentation patterns of these pyrrolizidine alkaloids were investigated, and 17 pyrrolidine alkaloids were identified. This approach could accelerate novel natural products discovery and characterize a class of natural products with MS/MS fragmentation patterns from similar chemical scaffolds. The research also provides a chemical basis for revealing in vivo effective substances in CP.