AbstractCurrent analytical protein methods show phosphorylation to be the most ubiquitous, evolutionary conserved post‐translational modification Post‐Translational Modification (PTM). The reversible and transient nature of protein phosphorylation allows signal transduction pathways to carry out diverse cellular functions. From bacteria to humans, phosphorylation serves to modify protein function by altering protein stability, cellular location, substrate affinity, complex formation, and activity; thus allowing essential events such as cell cycle and growth to occur at precise times and locations. Phosphorylation controls a variety of events at many biological levels including: housekeeping activities controlled by single cells such as DNA transcription, cell‐cycle regulation, and energy metabolism; and cellular processes that involve signaling between cells or the environment including such as neuronal migration and immune system recognition. This review summarizes state‐of‐the‐art proteomics technologies available to study phosphorylation in biological systems. We highlight the tremendous steps the field has made in the last 5 years which allow quantitative global analyses while pointing out caveats in experimentation. Copyright © 2009 John Wiley & Sons, Inc.This article is categorized under: Biological Mechanisms > Cell Signaling