Topoisomerases play a critical role in safeguarding the genome by resolving topological challenges that both result from and interfere with transcription and replication. These enzymes have been the subject of research not only for understanding their complex mechanisms of action, but also because of their therapeutic utility as drug targets. To gain a better understanding of topoisomerases and how they function, in vitro assays are necessary to measure their activity under various conditions. In this chapter, we describe the expression and purification of a eukaryotic type II topoisomerase (human topo IIα) from budding yeast cells and a gel-based assay that permits measurement of its ability to relax supercoiled DNA.