Full details of the development of a simple, nondestructive, and high-throughput method for establishing DNA binding affinity and sequence selectivity are described. The method is based on the loss of fluorescence derived from the displacement of ethidium bromide or thiazole orange from the DNA of interest or, in selected instances, the change in intrinsic fluorescence of a DNA binding agent itself and is applicable for assessing relative or absolute DNA binding affinities. Enlisting a library of hairpin deoxyoligonucleotides containing all five base pair (512 hairpins) or four base pair (136 hairpins) sequences displayed in a 96-well format, a compound's rank order binding to all possible sequences is generated, resulting in a high-resolution definition of its sequence selectivity using this fluorescent intercalator displacement (FID) assay. As such, the technique complements the use of footprinting or affinity cleavage for the establishment of DNA binding selectivity and provides the information at a higher resolution. The merged bar graphs generated by this rank order binding provide a qualitative way to compare, or profile, DNA binding affinity and selectivity. The 96-well format assay (512 hairpins) can be conducted at a minimal cost (presently ca. $100 for hairpin deoxyoligonucleotides/assay with ethiduim bromide or less with thiazole orange), with a rapid readout using a fluorescent plate reader (15 min), and is adaptable to automation (Tecan Genesis Workstation 100 robotic system). Its use in generating a profile of DNA binding selectivity for several agents including distamycin A, netropsin, DAPI, Hoechst 33258, and berenil is described. Techniques for establishing binding constants from quantitative titrations are compared, and recommendations are made for use of a Scatchard or curve fitting analysis of the titration binding curves as a reliable means to quantitate the binding affinity.