Delineating the precise regions on an antigen that are targeted by antibodies is important for the development of vaccines and antibody therapeutics. X-ray crystallography and NMR are considered the gold standard for providing precise information about these binding sites at atomic resolution. However, these are labor-intensive and require purified protein at high concentrations. We have recently described (Najar TA, Khare S, Pandey R, Structure 25:395–406, 2017) a rapid and reliable method that overcomes these constraints, using a panel of single cysteine mutants of the protein of interest, and now providing protocols to facilitate its adoption. Mutants are displayed on the yeast cell surface either individually or as a pool, and labeled covalently with a cysteine-specific probe. Binding site residues are inferred by monitoring loss of ligand or antibody binding by flow cytometry coupled to deep sequencing of sorted populations, or Sanger sequencing of individual clones. Buried cysteine residues are not labeled and library sizes are small, facilitating rapid identification of binding-site residues. The methodology was used to identify epitopes on the bacterial toxin CcdB targeted by 24 different monoclonal antibodies as well as by polyclonal sera. The method does not require purified protein or protein structural information and can be applied to a variety of display formats.