The MS fragmentation behavior of the C-21 steroidal glycosides from Dregea sinensis var. corrugata was investigated by positive and negative ion electrospray ionization MS using a multistage tandem mass spectrometer equipped with an ion trap analyzer. The mass fragmentation patterns of steroidal glycosides substituted with an orthoacetate group were summarized, and the fragmentation patterns were applied to the online structure identification of the steroidal glycosides in the extract. Eighteen new C-21 steroidal glycosides were identified by means of HPLC-HRESIMS and HPLC-DAD-ESIMS(n). Three new compounds (1, 4, and 7) were identified by HPLC-DAD-ESIMS(n), and their structures were elucidated by use of 1D and 2D NMR methods. The structures identified by MS are fully consistent with those elucidated by NMR data. The present study shows that HPLC-DAD-ESIMS(n) can be used as an effective tool to rapidly identify compounds and guide the isolation of target compounds from crude plant extracts.