DNA gyrase is an essential type II topoisomerase that is conserved across bacteria species and has an essential function of resolving overwound (positive supercoiled) DNA and introducing negative supercoils into relaxed DNA. The overall catalytic activity of gyrase can be determined using in vitro assays utilizing purified enzyme subunits and a DNA substrate. As gyrase is the only topoisomerase that can introduce negative supercoils into relaxed DNA, the inhibition of this catalytic activity is a good indicator of the efficacy and potency of potential antibacterial compounds. This chapter outlines a protocol for a purified enzyme assay with relaxed DNA that utilizes gel electrophoresis to monitor the ability of gyrase to introduce negative supercoils into DNA. The protocol focuses on the DNA supercoiling activity of Escherichia coli wild-type gyrase. However, it can be easily modified for use with gyrase from other bacterial species.