Knowledge of the exquisite binding surface of an antibody on its target protein is of great value, in particular for therapeutic antibodies for understanding the method of action and for stratification of patients carrying the necessary epitope for desired drug efficacy, but also for capture assays under native conditions. Several epitope mapping methodologies have been described for this purpose, with the laborious X-ray crystallography method being the ideal method for mapping discontinuous epitopes in antibody-antigen crystal complexes and high-throughput peptide-based methods for mapping linear epitopes. Here, we report on the usage of a bacterial surface display-based method for mapping structural epitopes by display of folded domains on the surface of Gram-positive bacteria, followed by domain-targeted mutagenesis and library analysis for identification of key-residues by flow sorting and sequencing. Identified clones with reduced affinity are validated by single clone FACS and subsequent full-length expression in mammalian cells for validation.