It is now well established that many therapeutic drugs exert their effects by binding to DNA primarily via intercalation and groove binding modes. It is therefore no surprise that this is an active area of study. Information on the exact DNA binding mode as well as the relative binding affinity have been instructive in understanding why and how these drugs bind to DNA. Drugs that bind to DNA via intercalation usually results in unwinding of the DNA molecule as the drug slides between adjacent base pairs. The extent to which unwinding occurs is usually drug-dependent. In this work, the relative DNA binding ability of a model homologous pair of naphthalene diimide intercalators were investigated using a topoisomerase I DNA unwinding assay. These compounds differ in substituent size and hydrophobicity. Also reported is a qualitative study of several well-known DNA binding compounds (netropsin, distamycin, berenil, Hoechst33258, DAPI) generally regarded as typical minor groove binding agents. The topoisomerase I DNA unwinding assay have revealed that some of these compounds exhibit unexpected modes of binding. The assay has also shown potential for determining the relative DNA binding affinity of homologous DNA intercalators. Binding affinity data for the naphthalene diimide intercalators was corroborated using isothermal titration calorimetry (ITC), spectrophotometric titration and DNA thermal melting (Tm) studies, with substituent size likely playing a significant role in binding. Future work will involve additional homologous members of the naphthalene diimide series as well as other series of intercalating compounds.