Protein phosphorylation pathways emerge as large and interconnected networks, involving mutually activating protein kinases, kinases acting as network nodes by phosphorylating different substrates, and cross-talk of phosphorylation with other post-translational modifications. The complexity of these networks clearly necessitates the use of systems biology approaches. Phosphoproteomics represents the basis for detection of phosphoproteins and phosphorylation sites, but it must be combined with transcriptomics and interactomics in attempts to build in silico phosphorylation networks. This review highlights the implication of phosphorylation in cellular physiology across all domains of life. It focuses particularly on reports of human disease correlated to defects in phosphorylation networks. Brief outline of developments in quantitative mass spectrometry-based proteomics and bioinformatic tools specific for phosphoproteome studies is provided.