Signaling pathways transduce extracellular stimuli from the membrane to the nucleus. Constitutive and thus inappropriate stimulation of these kinase cascades is associated with and observed in a majority of tumors. The transduction of signals in these pathways is achieved through protein-protein interactions regulated by changes in the phosphorylation status of key members. Therefore, the analysis of the interactions formed or broken in response to mitogenic stimulation is an important step toward understanding the molecular mechanisms of carcinogenesis. Today, mass spectrometry-based proteomics is one of the most widely used methods to unravel the molecular protein interaction networks that underlie these signaling cascades. This approach is powerful, but usually results in long lists of binding partners that may contain many false-positive hits and no information about the physiological role of the interacting proteins. Functional information can be derived by mapping changes in the interactome in response to specific stimuli or by comparing the interactome of related proteins with overlapping and different biological functions. As paradigms for these experimental approaches and the associated methodology, we describe here the functional proteomic analysis of the interactome of two distinct members of the mitogen-activated protein kinase (MAPK) cascade. The first is the analysis of interaction partners of the extracellular signal-regulated kinase (ERK) regulated by growth factor stimulation. The second is the differential analysis of binding partners of the C-terminal SH3 domain of the two small adaptor proteins Grb2 and GRAP.