Purified topoisomerases are used in a variety of in vitro assays to study their biology as well as their interactions with drugs. The purified enzymes can also be used in structural studies and as standards to quantify immunoblots. The one-step purification of His-tagged subunits from only 500 mL of expression culture described here yields topoisomerase enzymes that are pure enough and concentrated enough for in vitro activity assays and blotting standards.