The inhibition of DNA topoisomerase I (Top1) has proven to be a successful approach in the design of anticancer agents. However, despite the clinical successes of the camptothecin derivatives, a significant need for less toxic and more chemically stable Top1 inhibitors still persists. Here, we describe one of the most frequently used protocols to identify novel Top1 inhibitors. These methods use uniquely 3'-radiolabeled DNA substrates and denaturing polyacrylamide gel electrophoresis to provide evidence for the Top1-mediated DNA cleaving activity of potential Top1 inhibitors. These assays allow comparison of the effectiveness of different drugs in stabilizing the Top1-DNA intermediate or cleavage (cleavable) complex. A variation on these assays is also presented, which provides a suitable system for determining whether the inhibitor blocks the forward cleavage or religation reactions by measuring the reversibility of the drug-induced Top1-DNA cleavage complexes. This entire protocol can be completed in approximately 2 d.