A fully automated online multidimensional separation system，which is based on a reversed phase-strong cation exchange(RP-SCX) biphasic trap column，was developed for the large scale phosphorylation analysis of human liver.Compared with the conventional online SCX-RP system and off-line mode，the biphasic column can reduce the sample loss and improve the detection sensitivity.This system was applied in the large scale phosphorylation analysis of human liver and a total of 3 082 unique phosphopeptides，including 3 056 unique phosphorylation sites corresponding to 1 332 phosphoproteins were identified under the FDR＜1%.The molecular function and motif analysis of phosphoproteins has revealed the important role of phosphorylation in the liver physiological functions.