Type II DNA topoisomerases are essential, ubiquitous enzymes responsible for performing vital functions in chromosome condensation and segregation and in regulating intracellular DNA supercoiling. Topoisomerase II (topo II) performs these DNA transactions by passing one segment of DNA through the other using a reversible, enzyme-bridged double-stranded DNA break. This cleavage/religation of the DNA backbone is coupled to the opening and closing of the DNA gate, a critical step for strand passage during the catalytic cycle. To monitor the opening and closing of the DNA gate, we designed an oligonucleotide substrate with a pair of fluorophores flanking the topoisomerase II cleavage site, such that the fluorophores undergo efficient fluorescence resonance energy transfer (FRET) in the intact DNA substrate, but the FRET efficiency decreases as topo II opens the DNA gate. Here we present a method for creating the DNA substrate and using it as a tool to monitor the conformational changes at the topo II DNA gate. We detail how to collect and process fluorescence spectra to determine the FRET efficiency of the DNA substrate.