The present study was designed to develop a practical strategy to tackle the problem of lacking standard compounds and limited references for identifying structure-related compounds in Streptocaulon griffithii Hook.f.,especially those in trace concentrations,with a focus on antitumor activity.The cardiac glycosides (CGs)-enriched part was determined using in vitro bioactive assays in three cancer cell lines and then isolated using macroporous resins.The MS and MS/MS data were acquired using a high performance liquid chromatography coupled with hybrid quadrupole-time of flight (HPLC-Q-TOF-MS) system.To acquire data of trace compound in the extract,a multiple segment program was applied to modify the HPLC-Q-TOF-MS method.A mass defect filter (MDF) approach was employed to make a primary MS data filtration.Utilizing a MATLAB program,the redundant peaks obtained by imprecise MDF template calculated with limited references were excluded by fragment ion classification,which was based on the ion occurrence number in the MDF-filtered total ion chromatograms (TIC).Additionally,the complete cleavage pathways of CG aglycones were proposed to assist the structural identification of 29 common fragment ions (CFIs,ion occurrence number ≥ 5) and diagnostic fragment ions (DFIs,ion occurrence number ＜ 5).As a result,30 CGs were filtered out from the MDF results,among which 23 were identified.This newly developed strategy may provide a rapid and effective tool for identifying structure-related compounds in herbal medicines.