Characterization of post-translational modifications is one of the major challenges in proteomics. Reversible phosphorylation on serine, threonine and tyrosine residues is involved in regulation of many fundamental cellular events such as cell cycle, signal transduction, metabolism, and protein synthesis. To understand the molecular basis of these regulatory mechanisms, techniques capable of precise identification and quantitation of phosphorylation are needed. This review is devoted to recent trends in phosphoproteomics with special focus on mass spectrometry-based techniques. It covers enrichment of phosphoproteins/phosphopeptides using immobilized metal affinity chromatography and chemical derivatization techniques, phosphopeptide detection using differential peptide mass mapping, neutral loss and precursor ion scanning, localization of phosphorylation sites by tandem mass spectrometry as well as relative and absolute quantitation of phosphorylation stoichiometry.