Phospho-proteomics relies on methods for efficient purification and sequencing of phosphopeptides from highly complex biological systems using low amounts of starting material. Current methods for phosphopeptide enrichment, e.g., immobilized metal affinity chromatography and titanium dioxide chromatography, provide varying degrees of selectivity and specificity for phosphopeptide enrichment. Furthermore, the number of multiply phosphorylated peptides that are identified in most published studies is rather low. Here the protocol for a new strategy that separates mono-phosphorylated pep-tides from multiply phosphorylated peptides using sequential elution from immobilized metal affinity chromatography is described. The two separate phosphopeptide fractions are subsequently analyzed by mass spectrometric methods optimized for mono-phosphorylated and multiply phosphorylated peptides, respectively, resulting in improved identification of especially multiply phosphorylated peptides from a minimum amount of starting material. The new method increases the coverage of the phosphoproteome significantly.