The development of antibodies for either detection or therapeutics requires extensive characterization of several potential leads. One of the critical steps in the characterization process is epitope mapping (binning), where the different antibodies are divided into “bins” according to their antigen recognition site, which is directly linked to their functional activity. Biolayer interferometry (BLI) is a technology used to characterize interactions between macromolecules and, as such, is used extensively to characterize antibodies. The current protocol describes the use of BLI for antibody binning. Along the classical pairwise mapping model, this protocol addresses the challenges of binning on multivalent antigens as well as characterizing non-purified antibodies.