Cells are highly responsive to their environment. One of the main strategies used by cells in signal transduction is protein phosphorylation, a reversible modification that regulates numerous biological processes. Misregulation of phosphorylation-mediated processes is often implicated in many human diseases and cancers. A global and quantitative analysis of protein phosphorylation provides a powerful new approach and has the potential to reveal new insights in signaling pathways. Recent technological advances in high resolution mass spectrometers and multidimensional liquid chromatography, combined with the use of stable isotope labeling of proteins, have led to the application of quantitative phosphoproteomics to study in vivo signal transduction events on a proteome-wide scale. Here we review recent advancements in quantitative phosphoproteomic technologies, discuss their potentials and identify areas for future development. A key objective of proteomic technology is its application to addressing biological questions. We will therefore describe how current quantitative phosphoproteomic technology can be used to study the molecular basis of phosphorylation events in the DNA damage response.