A new and systematic application for separation and online identification of coumarins from Peucedanum praeruptorum Dunn by preparative high-speed counter-current chromatography coupled with electrospray ionization multi-stage mass spectrometry (prep-HSCCC/ESI-MS(n)) was established. The procedure of separation was guided by the chromatogram of ion current. The structures of acquisitions were deduced by MS information. The hyphenation between prep-HSCCC/ESI-MS(n) was designed to keep the split ratio from 1:20 to 1:200 exactly. Seven compounds were obtained and two new compounds were detected. It was proved that prep-HSCCC/ESI-MS(n) was an effective method for sensitive detection, rapid identification and separation of natural products.