Objective: Quality control of traditional Chinese medicine (TCM) begins with the chemical basis elucidation. The root of Stephania tetrandra has long been utilized as an antirheumatic, analgesic, and diuretic TCM, Stephaniae Tetrandrae Radix (STR; Fang-Ji). Powerful analytical strategies enabling its multicomponent characterization is still rare. Methods: A rapid, reliable, and enhanced profiling approach, by ultra-high performance liquid chromatography coupled with ion mobility/quadrupole time-of-flight mass spectrometry (UHPLC/IM-QTOF-MS) and automatic peak annotation facilitated by computational matching of in-house library, was established and utilized to characterize the multicomponents from STR. A knockout strategy was utilized by automated valve switching to overcome the interference of predominant peaks. Results: Good chromatographic separation was achieved within 17 min on a reversed-phase BEH C18 column eluted with acetonitrile/0.1% ammonium hydroxide in water, while data-independent high-definition MSE (HDMSE) in positive mode was applied to acquire the MS2 data by using a VionTM IM-QTOF instrument, which in theory, could cover all the profiled precursor ions. An in-house library of 163 compounds was established and incorporated into the UNIFITM platform. By feat of these efforts, we were able to identify or tentatively characterize 76 alkaloids from the methanolic extract of STR, including 14 aporphine-type, four morphine-type, 48 bisbenzylisoquinoline-type, seven tetrahydroprotoberberine-type, one protopine-type, one benzylisoquinoline-type, and one other. Four-dimensional information, such as the retention time, collision cross section (CCS), high-accuracy MS1 and MS2 data, for each component was provided. Conclusions: The systematic multicomponent characterization of STR was accomplished with high coverage, high degree of automation, and high reliability.