The MUC4 membrane-bound mucin is a large O-glycoprotein involved in epithelial homeostasis. At the cancer cell surface MUC4 interacts with ErbB2 receptor via EGF domains to promote cell proliferation and migration. MUC4 is highly regarded as a therapeutic target in pancreatic cancer as it is not expressed in healthy pancreas, while it is neoexpressed in early preneoplastic stages (PanINs). However, the association/dissociation constant of MUC4-ErbB2 complex is unknown. Protein-protein interactions (PPIs) have become a major area of research in the past years and the characterization of their interactions, especially by biophysical methods, is intensively used in drug discovery. To characterize the MUC4-ErbB2 interaction, we used MicroScale Thermophoresis (MST), a powerful method for quantitative protein interaction analysis under challenging conditions. We worked with CHO cell lysates containing either the transmembrane β subunit of MUC4 (MUC4β) or a truncated mutant encompassing only the EGF domains (MUC4 EGF3+1+2). MST studies have led to the characterization of equilibrium dissociation constants (K d) for MUC4β-ErbB2 (7-25 nM) and MUC4 EGF3+1+2 /ErbB2 (65-79 nM) complexes. This work provides new information regarding the MUC4-ErbB2 interaction at the biophysical level and also confirms that the presence of the three EGF domains of MUC4 is sufficient to provide efficient interaction. This technological approach will be very useful in the future to validate small molecule binding affinities targeting MUC4-ErbB2 complex for drug discovery development in cancer. It will also be of high interest for the other known membrane mucins forming oncogenic complexes with ErbBs at the cancer cell surface. Context Mucins form a family of large O-glycoproteins, of heterogeneous evolutionary origin, organized into a peptidic chain called apomucin in which a Serine/Threonine-rich region is intensively glycosylated. In addition to this O-glycosylation, which represents up to 50-80% of the total molecular weight of the protein, N-glycosylation may be present to a lesser extent 1. MUC4 belongs to the membrane-bound mucin class and is synthesized as a single chain that may be cleaved into two subunits tightly associated by non-covalent interactions. MUC4α is the extracellular mucin-like O-glycosylated subunit whereas MUC4β is the membrane-tethered subunit. MUC4β contains several functional domains, such as epidermal growth factor (EGF)-like domains, Von Willebrand factor type D (VWD) domain and a single transmembrane helix (TM) with a short cytoplasmic tail (CT) 2 (Fig. 1). Membrane-bound mucins have been reported to be involved in pathological disorders and particularly in neo-plastic development of cancers 3,4. MUC4 is indeed extensively regarded as an overexpressed pro-tumorigenic protein in epithelial cancers (such as lung, esophagus, colon, breast or pancreas 5) as it forms an oncogenic complex with ErbB2 receptor tyrosine kinase. The physical interaction between human MUC4 and ErbB2 involves a region of the extracellular domain of MUC4β composed of three EGF-like domains 6. Two of the three EGF-like domains (EGF1 and EGF2), conserved throughout evolution, are structurally equivalent to human EGF and biologically active 7-10. Moreover,