Protein phosphorylation is the best‐studied posttranslational modification and plays a role in virtually every biological process. Phosphoproteomics is the analysis of protein phosphorylation on a proteome‐wide scale, and mainly uses the same instrumentation and analogous strategies as conventional mass spectrometry (MS)‐based proteomics. Measurements can be performed either in a discovery‐type, also known as shotgun mode, or in a targeted manner which monitors a set of a priori known phosphopeptides, such as members of a signal transduction pathway, across biological samples. Here, we delineate the different experimental levels at which measures can be taken to optimize the scope, reliability, and information content of phosphoproteomic analyses. Various chromatographic and chemical protocols exist to physically enrich phosphopeptides from proteolytic digests of biological samples. Subsequent mass spectrometric analysis revolves around peptide ion fragmentation to generate sequence information and identify the backbone sequence of phosphopeptides as well as the phosphate group attachment site(s), and different modes of fragmentation like collision‐induced dissociation (CID), elect
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