In this paper, we describe the technique of analytical highspeed countercurrent chromatography coupled with electrospray ionization mass spectrometry (HSCCC/ESIMS) for the first time, and evaluate its performance in terms of chromatography and mass spectrometry. This technique demonstrates the capability and resolving power of HSCCC in the separation of a few natural product mixtures and utilizes ESIMS as a detector with continuous detection and high sensitivity. Application of this HSCCC/ESIMS system is illustrated by the separation and detection of biologically important compounds.