New analytical strategies for phosphoproteomics, both experimental and computational, have been rapidly introduced in recent years, leading to novel biological findings on the role of protein phosphorylation, which have in turn stimulated further development of the analytical techniques. In this review, we describe the development of analytical strategies for LC-MS/MS-based phosphoproteomics, focusing particularly on recent progress in phosphopeptide enrichment, LC-MS/MS measurement and the subsequent computational analysis. High-coverage analysis of the phosphoproteome has largely been achieved by combining pre-fractionation methods with multiple phosphopeptide enrichment approaches, at some cost in LC-MS/MS measurement time and increased sample loss. Key points for the future will be to further increase the selectivity and the recovery of enrichment methods to achieve higher sensitivity and efficiency in LC-MS/MS analysis in order to detect protein phosphorylation comprehensively, including low-abundance proteins. This is expected to lead to a more detailed understanding of the mechanisms and interactions of phosphorylation-mediated regulatory pathways in biological systems. (c) 2012 Elsevier Ltd. All rights reserved.