Phosphorylation of proteins plays an important role of post-translational modifications in signal pathways and cellular metabolism. However, the difficulty lies in identification of phosphorylated proteins and phosphorylation site due to its low abundance in cells. The novel enrichment methods of phosphopeptides are of keen interest prior to the identification. Already we have developed the high sensitive and high throughput protein profiling system, micro-LC/NSI-ITMS (micro-flow liquid chromatography/ nanoelectronspray ion-trap mass spectrometry) designed to be metal ion free. In addition, data dependent neutral loss scan to trigger MS3 fragments is investigated. Therefore, we have investigated the construction of phosphorylated proteins profiling system which integrates enrichment methods of phosphopeptides and multi-dimensional LC/MS. Here, we will present the integration of the above techniques for phosphopeptides analysis and its application.