Phosphorylation is one of the most frequently occurring post-translational modifications in proteins. In eukaryotic cells, protein phosphorylation on serine, threonine and tyrosine residues plays a crucial role as a modulator of protein function. A comprehensive analysis of protein phosphorylation involves the identification of the phosphoproteins, the exact localization of the residues that are phosphorylated and the quantitation of phosphorylation. In this short review we will summarize and discuss the methodologies currently available for the analysis and full characterization of phosphoproteins with special attention at mass spectrometry-based techniques. In particular, we will discuss affinity-based purification of phosphopeptides coupled to MALDI-TOF analysis, their detection using mass mapping and precursor ion scan, identification of modified sites by MS/MS and quantitation analysis