The diversity and complexity of proteins and peptides in biological systems requires powerful liquid-chromatography based separations to optimize resolution and detection of components. Proteomics strategies often combine two orthogonal separation modes to meet this challenge. In nearly all cases, the second dimension is a reverse phase separation interfaced directly to a mass spectrometer. Here we report on the use of Hydrophilic-Interaction Chromatography (HILIC) as part of a multidimensional chromatography strategy for proteomics. Tryptic peptides are separated on TSKgel Amide-80 columns using a shallow inverse organic gradient. Under these conditions, peptide retention is based on overall hydrophilicity and a separation truly orthogonal to reverse phase is produced. Analysis of tryptic digests from HeLa cells yield comparable numbers of protein identifications to that obtained using strong cation exchange. We also demonstrate that HILIC represents a significant advance in phosphoproteomic analysis. We exploited the strong hydrophilicity of the phosphate group to selectively enrich and fractionate phosphopeptides based on their increased retention under HILIC conditions. Subsequent IMAC enrichment of phosphopeptides from HILIC fractions show better than 99% selectivity. This is achieved without the use of derivatization or chemical modifiers. In a 300ug equivalent of HeLa cell lysate we identified over 1000 unique phosphorylation sites. More than 700 novel sites are added to the HeLa phosphoproteome.