Hydrophilic interaction liquid chromatography(HILIC) was selected to separate glucosides according to their structure characteristics.The insufficient of selectivity of reversed-phase chromatography(RPLC) would be made up.Firstly,the separation effects of 14 glucosides by both HILIC and RPLC columns were compared.The orthogonality was evaluated.Then the 2-D RPLC/HILIC system was used to separate American ginseng extract.The results showed that the glucosides were well reserved on both HILIC and RPLC columns.But the sequence of peaks was very different.Ginsenoside Rg1 and Re could not be separated with RPLC column,but they could be separated absolutely with HILIC column.The RPLC and HILIC modes had a high orthogonality.The peak capacity and separation ability were well increased for analyzing American ginseng extract by this 2-D RPLC/HILIC system.It was beneficial to the discovery of new compounds.This method was simple,and the mobile phase was compatible with both chromatographic modes.It could be used as the effective means for separation and preparation of glucosides.Also,it could provide a reference for the analysis of other complex traditional Chinese medicines.