Abstract: Protein phosphorylation is a post-translational modification widely used to regulate cellular responses. Recent studies showed that global phosphorylation analysis could be used to study signaling pathways and to identify targets of protein kinases in cells. A key objective of global phosphorylation analysis is to obtain an in-depth mapping of low abundance protein phosphorylation in cells, which necessitates the use of suitable separation techniques due to the complexity of the phosphoproteome. Here we developed a multidimensional chromatography technology, combining Immobilized Metal Affinity Chromatography (IMAC), Hydrophilic Interaction Chromatography (HILIC) and Reverse-Phase Liquid Chromatography (RP-LC), for phosphopeptide purification and fractionation. Its application to the yeast S. cerevisiae after DNA damage led to the identification of 8,764 unique phosphopeptides from 2,278 phosphoproteins using tandem mass spectrometry (MS). Analysis of two low abundance proteins, Rad9 and Mrc1, revealed that approximately 50 % of their phosphorylation was identified via this global phosphorylation analysis. Thus, this technology is suited for in-depth phosphoproteome studies.