The proteins that interact with DNA to maintain fidelity of DNA synthesis, replication, and balanced gene expression within healthy cells can, under certain circumstances, become mediators of disease. Although these interactions are quite complex, recent advances in proteomics and genomics have identified many DNA-binding proteins and their DNA target sequences. Historically, binding affinities between protein and DNA have been measured using the gel mobility shift assay (GMSA). This assay is time consuming, involves use of radioactivity, and is not a true equilibrium measurement because of the gel matrix involved. With the recent advancements in synthesis of fluorochrome-labeled oligonucleotides and the advent of more sophisticated fluorescent plate-readers, fluorescence anisotropy offers an excellent alternative to GMSA. By placing the fluorescent signal on the smaller DNA molecule, binding to the much larger protein results in a substantial change in anisotropy and enables detection of binding constants within the range of 10-10 to 10-3 molar. We provide a description of a solution-based methodology to determine protein-DNA binding, dissociation and competition parameters under either equilibrium or kinetic conditions with use of minimal volumes and short assay times.