The maintenance of DNA topology is critical for cellular processes. Topoisomerases are a class of enzymes found in eukaryotic and prokaryotic organisms that modulate the topology of DNA during replication and transcription. Bacterial type II topoisomerases, DNA gyrase and topoisomerase IV, are essential for bacterial cell survival. These enzymes pass an intact double helix through a transient double-stranded break in a separate segment of DNA in order to carry out their specific functions. The DNA cleavage step is crucial for the activity of the type II enzymes. However, prolongation of this process, for example, with exposure to antibacterial agents, can lead to bacterial cell death. The DNA cleavage assay is an in vitro method used to study the effects of type II topoisomerase-targeting compounds on enzyme-mediated DNA cleavage. This chapter outlines a protocol for measuring DNA cleavage mediated by Escherichia coli gyrase in the presence or absence of antibacterial drugs.