Protein phosphorylation represents one of the most extensively studied post-translational modifications, primarily due to the emergence of sensitive methods enabling the detection of this modification both in vitro and in vivo. The availability of enrichment methods combined with sensitive mass spectrometry instrumentation has played a crucial role in uncovering the dynamic changes and the large expanding repertoire of this reversible modification. The structural changes imparted by the phosphorylation of specific residues afford exquisite mechanisms for the regulation of protein functions by modulating new binding sites on scaffold proteins or by abrogating protein–protein interactions. However, the dynamic interplay of protein phosphorylation is not occurring randomly within the cell but is rather finely orchestrated by specific kinases and phosphatases that are unevenly distributed across subcellular compartments. This spatial separation not only regulates protein phosphorylation but can also control the activity of other enzymes and the transfer of other post-translational modifications. While numerous large-scale phosphoproteomics studies highlighted the extent and diversity of phosphoproteins present in total cell lysates, the further understanding of their regulation and biological activities require a spatio-temporal resolution only achievable through subcellular fractionation. This review presents a first account of the emerging field of subcellular phosphoproteomics where cell fractionation approaches are combined with sensitive mass spectrometry methods to facilitate the identification of low abundance proteins and to unravel the intricate regulation of protein phosphorylation. © 2010 Wiley Periodicals, Inc. Mass Spec Rev 29:962–990, 2010