Posttranslational modifications of proteins are relevant for a large variety of cellular functions. We constantly optimize protocols to specifically enrich and analyze phosphorylated peptides from tissue or cell lysates, in order to identify alterations in phosphorylation-dependent signal transduction pathways comparing treated to untreated cellular conditions. In our approach, a total proteome sample as well as a phosphorylation-enriched peptide fraction are generated from the same sample in a two-step protocol. This enables normalization and evaluation of changes on the phosphorylation level in comparison to the total proteome. Samples are analyzed by data dependent acquisition (DDA) or data independent acquisition (DIA) followed by label-free quantification. Meaningful phosproteome profiling requires prior experimental knowledge about the dynamics (chronic or transient effects on phosphorylation) and the timing of the respective stimulation.