DNA topoisomerase II (TOP2) regulates DNA topological states including supercoiling and knotting via a strand-passage reaction that involves transiently breaking and rejoining both strands of the DNA. These enzymes are the targets of a category of drugs termed topoisomerase poisons, which block the rejoining reaction, leading to DNA cleavage. In vitro DNA cleavage assays have been very useful in studying various properties of TOP2, including cleavage site preferences and the effects of TOP2 amino acid substitutions or reaction conditions on DNA cleavage. These assays employ purified or recombinant TOP2 in the presence or absence of TOP2 poisons, followed by electrophoretic separation of DNA cleavage products. The substrates for these assays have typically been radioactively end-labelled DNA fragments. Here, we describe an alternative method employing fluorescently labelled oligonucleotide substrates, combined with convenient mini-gel electrophoretic separation. This methodology combines stable, long-lived substrates with an easily used gel system and convenient imaging and quantification.