In 1980, it was found that epidermal growth factor (EGF) stimulated the phosphorylation of proteins on tyrosine residues. Subsequently, work from a number of different laboratories indicated that in addition to EGF, platelet-derived growth factor (PDGF), insulin, and insulin-like growth factor I (IGF-I) stimulated the phosphorylation of proteins on tyrosine residues. The data suggested that each of these growth factor receptors possessed intrinsic tyrosine kinase activity that was regulated by the growth factor. These tyrosine kinase activities were first identified by the ability of these kinases to carry out an autophosphorylation reaction. While the use of the growth factor receptors themselves as specific substrates for these kinases was helpful in determining the relationship between the receptor and the kinase, the lack of a suitable exogenous substrate limited the types of experimental questions that could be approached. This chapter discusses the assay method of the growth factor-stimulated tyrosine kinases with the synthetic peptide. The assay of the growth factor-stimulated tyrosine kinases is based on their ability to catalyze the transfer of the γ-phosphate of ATP to a suitable acceptor substrate, which is a synthetic, tyrosine-containing peptide.