Antibodies play crucial roles in both basic research and clinical practice. The binding interface between an antibody and its antigen, i.e., the epitope, is a defining feature of the antibody. Accurate epitope mapping is essential for developing antibody-based reagents and therapies and safeguarding intellectual property. Here, we present the detailed procedure of a novel high-throughput technology, namely, Antibody binding epitope Mapping (AbMap) version 1.0. Leveraging versatile phage-displayed peptide library and next-generation sequencing (NGS), AbMap enables the comprehensive profiling of hundreds to thousands of antibody-binding epitopes in a single run.