This study presents the novel direct coupling of preparative high-speed counter-current chromatography with electrospray ionization mass spectrometry (HSCCC/ESI-MS-MS) for continuous MS data acquisition, and 'target'-guided fractionation of polyphenolic compounds from sea buckthorn juice concentrate (Hippophaë rhamnoides L. ssp. rhamnoides, Elaeagnaceae). It was demonstrated that a combination of HSCCC and ESI-MS-MS is a versatile tool for fast screening and sensitive detection of bioactive natural products in crude extracts. Sample fractionation will be simplified by knowing the continuous MS data of the complete HSCCC separation. Already during the separation, immediate structural information of the separated compounds is provided. Peak purity of HSCCC is assessable by complete ion traces and fractions can directly be evaporated for further one-/two-dimensional NMR analysis.