AbstractProtein tyrosine kinases (PTKs) are ubiquitous enzymes that are integrally involved in the regulation of transformation mechanisms, normal and pathological growth, cell cycle regulation, immune responses, and a variety of intracellular signaling mechanisms. This rapidly growing family of enzymes is generally divided into two groups: receptor PTKs (with more than twelve distinct families) and nonreceptor PTKs (with more than nine distinct families). PTKs mediate the enzymatic transfer of the gamma phosphate of ATP to the phenolic groups on tyrosine residues to generate phosphate monoesters. In this unit, several assays are provided to measure the ability of PTKs to transphosphorylate protein and peptide substrates, and to autophosphorylate. Phosphorylation of exogenous substrates or autophosphorylation is detected using a 32P‐ or 33P‐phosphorylated protein. Alternatively, antibodies recognizing phosphorylated tyrosine residues can be used to quantify PTK activity. In some cases, antibodies are available for context‐specific phosphotyrosine residues, thereby enabling the detection of PTK‐specific substrate phosphorylation.