Cells sense and respond to mitogens by activating a cascade of signaling events, primarily mediated by tyrosine phosphorylation (pY). Because of its key roles in cellular homeostasis, deregulation of this signaling is often linked to oncogenesis. To understand the mechanisms underlying these signaling pathway aberrations, it is necessary to quantify tyrosine phosphorylation on a global scale in cancer cell models. However, the majority of the protein phosphorylation events occur on serine (86%) and threonine (12%) residues, whereas only 2% of phosphorylation events occur on tyrosine residues (Olsen et al., 2006). The low stoichiometry of tyrosine phosphorylation renders it difficult to quantify cellular pY events comprehensively with high mass accuracy and reproducibility. Here, we describe a detailed protocol for isolating and quantifying tyrosine phosphorylated peptides from drug-perturbed, growth factor-stimulated cancer cells, using immunoaffinity purification and tandem mass tags (TMT) coupled with mass spectrometry.