Gaining a better understanding of normal cellular signaling and its modulation in disease is a critical goal of mass spectrometry-based proteomic analyses. In particular, measurement of changes in post-translational modifications (PTMs), such as phosphorylation, is a key metric for elucidating the activity of signaling pathways and changes in activity post-treatment. Many successful studies have utilized the power of mass spectrometry to provide insights into these changes, including projects that have determined novel sites of protein phosphorylation, ubiquitination, acylation, methylation, or protease cleavage; identified and validated drug targets; discovered biomarkers; explored the mechanism of action of drugs/chemical modulators; and elucidated off-target drug effects. In all of these cases, the enrichment of the PTM of interest was the key step to allow these discoveries to be made. Without enrichment, the signal of PTM peptides is lost in the background of all other, unmodified peptides in a cell line, tissue, or biofluid sample.