Fluorescence spectroscopy titrations, although widely used to analyze binding affinity, are not an efficient screening method for detecting high-affinity binding among a large number of available ligands, such as during bacteriophage display selections. We hypothesize that a miniaturized, high-throughput fluorescence spectroscopy assay can be used to efficiently analyze selection results by applying the Langmuir equation to the binding data to estimate affinity constants for a large number of ligands, either as synthesized molecules or as displayed on bacteriophage. Here, bacteriophage-display-derived peptides specific for the Thomsen-Friedenreich disaccharide are used to develop a high-throughput fluorescence spectroscopy screening method, which uses one binding partner labeled with a fluorescent dye and different concentrations of a second partner to analyze binding affinity in bacteriophage display selections. The affinity constants derived from binding isotherms prepared using the new system accurately replicate those derived from standard spectroscopy titrations. Furthermore, the technique correctly defined the affinity constant describing binding of a cognate epitope peptide by a monoclonal antibody. Finally, we have applied the technique to analysis of binding affinity by ligands displayed on bacteriophage, which suggests that this technique could be used to monitor bacteriophage enrichment during selections.