The identification of protein phosphorylation sites from cell-derived proteins is crucial to the understanding of signal transduction pathways. While determining the modified sites on individual proteins can present a significant challenge, recent progress in the rapid, large-scale identification of phosphopeptides from complex protein mixtures by combinations of affinity chromatography and mass spectrometry provides a powerful tool to decipher the phosphoproteome. A set of protocols is described for sample preparation, fractionation, immobilized metal ion affinity chromatography (IMAC), and mass spectrometric analysis of phosphorylation sites. Parts of the protocols can be combined in different ways to adapt to sample amounts, complexity, and available equipment. Up to thousands of unique phosphopeptides can be sequenced from a single sample in a day, revealing a unique snapshot of global cellular phosphorylation sites on proteins and facilitating in-depth study of the identified phosphoproteins.