PTMs enable cells to adapt to internal and external stimuli in the milliseconds to seconds time regime. Protein phosphorylation is probably the most important of these modifications as it affects protein structure and interactions, critically influencing the life cycle of a cell. In the last 15 years, new insights into phosphorylation have been provided by highly sensitive MS-based approaches combined with specific phosphopeptide enrichment strategies. Although so far research has mainly focused on the discovery and characterization of O-phosphorylation, this review also briefly outlines the current knowledge about N-phosphorylation depicting its ubiquitous relevance. Further, common pitfalls in sample preparation, LC-MS analysis, and subsequent data analysis are discussed as well as issues regarding quality and comparability of studies on protein phosphorylation.