Two phenylethanoid glycosides(PhGs), plantamajoside and acteoside, were isolated and purified from the aerial parts of Plantago asiatica for the first time by high performance centrifugal partition chromatography(HPCPC) with ethyl acetate, n-butanol, ethanol and water(0.5:0.5:0.1:1, volume ratio) as solvent system. A total of 45.6 mg of plantamajoside and 293.8 mg of acteoside were purified from 1341 mg of the n-butanol extract of P. asiatica, with a purity of >93.3% as determined by HPLC. The HPCPC fractions were analyzed by HPLC-DAD and the structures were identified by their retention time, UV, electrospray ionization multi stage tandem mass spectrometry(ESI-MSn) in the negative ion mode, and confirmed by NMR experiments. The characteristic fragment ions of ESI-MS of the two PhGs isolated from Plantago asiatica were discussed, which are specific and useful for the identification of the structures of PhGs. Two phenylethanoid glycosides (PhGs), plantamajoside and acteoside, were isolated and purified from the aerial parts of Plantago asiatica for the first time by high performance centrifugal partition chromatography (HPCPC) with ethyl acetate, n-butanol, ethanol and water A total of 45.6 mg of plantamajoside and 293.8 mg of acteoside were purified from 1341 mg of the n-butanol extract of P. asiatica, with a purity of> 93.3% as determined by HPLC. The HPCPC fractions were identified by HPLC-DAD and the structures were identified by their retention time, UV, electrospray ionization multi-stage tandem mass spectrometry (ESI-MSn) in the negative ion mode, and confirmed by NMR experiments. The characteristic fragment ions of ESI-MS of the two PhGs isolated from Plantago asiatica were discussed, which are specific and useful for the identification of the structures of PhGs.