A novel, high-throughput-compatible assay for the ATP-dependent supercoiled DNA relaxing activity of human topoisomerase IIα (hTopoIIα) is described. The principle of detection is the preferential binding of the oligodeoxyribonucleotide BODIPY-TMR-5′-TTCTTCTTCT-3′ to relaxed double-stranded plasmid containing the triplex forming sequence (TTC)9 versus the supercoiled plasmid. Binding of the oligonucleotide to the plasmid increases the fluorescence anisotropy of the BODIPY-TMR label. Optimization of the assay conditions was conducted to maximize the signal and the activity of the topoisomerase. The multiwell assay plate-based fluorescence anisotropy assay gave the same values for the potencies of several previously reported inhibitors of hTopoIIα as a gel electrophoresis-based assay of DNA relaxation.