In order to study receptor abundance and its function in solutions or in homogenates from clinical specimen, methods such as sandwich or radioimmunoassays are most commonly employed. For the determination of epidermal growth factor receptor (EGFR), we describe the development of a miniaturized bead-based ligand binding assay using its ligand EGF as immobilized capture reagent. This assay was used to analyze lysates from cell lines, and the ligand-bound EGFR was detected using an EGFR-specific antibody combined with a fluorescence-based reporter system. In a proof-of concept study with lysates from breast biopsies, the assay allowed to classify breast cancer samples in accordance to clinically the relevant EGFR cut-off level. The study suggests that such a ligand binding receptor assay could become an integral part of protein profiling procedures to provide additional information about receptor functionality in addition to its abundance.