Aim:To develop a robust,cell-based,high-content screening (HCS) assay basedon receptor internalization for the identification of novel modulators of the epi-dermal growth factor receptor (EGFR).Methods:Agonist-induced receptorinternalization is part of the signaling cascade of EGFR.Fluorescent-taggedepidermal growth factor (EGF) was used to visualize the internalized receptor-ligand complex.The fluorescent intracellular spots were detected and mea-sured with an ArrayScan HCS reader.Compounds that can competitively bind toEGFR or interfere with EGFR internalization process would result in a reducednumber and intensity of intracellular fluorescent spots.This assay was validated,optimized,and applied to a large-scale screening of a library containing 48 000synthetic compounds.Results:The competition between fluorescent EGF andunlabeled EGF reveals the IC_(50) of unlabeled EGF is approximately 0.2 nmol/L,which is comparable with other published reports.Thirteen compounds with arelatively high degree of interference with EGFR internalization were identified.One of the compounds was proven to be agonist of the EGFR since it inducedphosphorylation of the receptor and extracellular signal-regulated protein ki-nase (ERK).Conclusion:This automated,objective,and easy-to-use assay pro-vided abundant information,quantitative results,and demonstrated the potentialuse of HCS methods in searching membrane receptor modulators. Aim: To develop a robust, cell-based, high-content screening (HCS) assay based on receptor internalization for the identification of novel modulators of the epi-dermal growth factor receptor (EGFR). Methods: Agonist-induced receptor internalization is part of the signaling cascade of EGFR. Fluorescent-taggedepidermal growth factor (EGF) was used to visualize the internalized receptor-ligand complex. The fluorescent intracellular spots were detected and mea- sured with an ArrayScan HCS reader. Compounds that can competitively bind to EGFR or interfere with EGFR internalization process would result in a reduced number and intensity of intracellular fluorescent spots. This assay was validated, optimized, and applied to a large-scale screening of a library containing 48 000 synthetic compounds. Results: The competition between fluorescent EGF and uncomlabeled EGF reveals the IC_ ( 50) of unlabeled EGF is approximately 0.2 nmol / L, which is comparable with other published reports. Thirteen compounds with arelatively h igh degree of interference with EGFR internalization identified. One of the compounds was proven to be agonist of the EGFR since it induced phosphorylation of the receptor and extracellular signal-regulated protein ki-nase (ERK). Confusion: This automated, objective, and easy -to-use assay pro-vided abundant information, quantitative results, and demonstrated the potentialuse of HCS methods in searching membrane receptor modulators.