The complete analysis of phosphoproteomes has been hampered by the lack of methods for efficient purification, detection and characterization of phosphorylated peptides from complex biological samples. Despite several strategies for affinity enrichment of phosphoryated peptides prior to mass spectrometric analysis, such as immobilized metal affinity chromatography or titanium dioxide the coverage of the phosphoproteome of a given sample is limited. Here we report a simple and rapid strategy – SIMAC - for sequential separation of mono-phosphorylated peptides and multiply phosphorylated peptides from highly complex biological samples. This allows individual analysis of the two pools of phosphorylated peptides using mass spectrometric parameters differentially optimized for their unique properties. We compared the phosphoproteome identified from 120 µg of human mesenchymal stem cells using SIMAC and an optimized titanium dioxide chromatographic method. More than double the total number of identified phosphorylation sites was obtained with SIMAC, primarily from a three-fold increase in recovery of multiply phosphorylated peptides.