Updated publication reference for PubMed record(s): 29535314. Comprehensive mass spectrometry (MS)-based proteomics is now feasible, but reproducible and multiplexed quantification remains challenging especially for analysis of post-translational modifications (PTMs), such as phosphorylation. Here we compared the most popular quantification techniques for phosphoproteomics in context of cell-signaling studies: label-free quantification (LFQ), stable isotope labeling by amino acids in cell culture (SILAC) and MS2- and MS3-measured tandem mass tags (TMT). In a mixed species comparison with fixed phosphopeptide-ratios, we found LFQ and SILAC to be the most accurate techniques. MS2-based TMT suffered from substantial ratio compression, which MS3-based TMT could partly rescue. However, when analyzing phosphoproteome changes in the DNA damage response (DDR), we found that MS3-based TMT was outperformed by MS2-based TMT as it identified most significantly regulated phosphopeptides due to its higher precision and higher number of identifications. Finally, we show that the high accuracy of MS3-based TMT is crucial for determination of phosphorylation site stoichiometry using a novel multiplexing-dependent algorithm.