Magnetic tweezers provide a versatile tool enabling the precise application of force and torque on -individual biomolecules. These properties make magnetic tweezers uniquely suited for the study of DNA topology and topoisomerases at the single-molecule level. Single-molecule approaches, which are complementary to ensemble biochemical and structural approaches, have provided remarkable insights into the mechanisms of topoisomerase activity and interactions with DNA. Here, we describe how to make single-molecule measurements of topoisomerase activity with a magnetic tweezers instrument. We provide detailed instructions for preparing and characterizing DNA substrates, flow cells, and supercoilable DNA tethers. We then describe magnetic tweezers measurements of supercoil relaxation by single topoisomerases.