Protein phosphorylation is crucial in the regulation of signaling pathways that 
control various biological responses. Recent progress in diverse methodologies 
to investigate protein phosphorylation in complex biological samples has 
resulted in more rapid, detailed and quantitative analyses of signaling 
networks. In particular, advances in mass spectrometry (MS) have enabled the 
identification and quantification of thousands of both known and novel 
phosphorylation sites. Initial MS-based information can be complemented with a 
variety of recently developed and improved phosphoproteomic techniques. These 
include multiplexed microbead or kinase activity assays, flow cytometry based 
single-cell analysis, protein microarrays and interaction studies. The 
combination of multiple approaches, coupled with phenotypic response 
measurements, computational modeling and biochemical manipulations, will 
ultimately reveal the mechanistic regulation of signaling networks.