Topoisomerases unlink chromosomes and relieve topological stress. Topoisomerase “poisons” are widely used chemotherapeutics that stabilize topoisomerase complexes on DNA, leading to cytotoxic DNA breaks and cancer cell killing. It is well established that topoisomerase poisons interfere with DNA replication, which is thought to be a major physiological target of these drugs. However, many questions remain about the mechanisms by which topoisomerase poisons impact DNA replication and the downstream consequences. Here, we describe assays to study topoisomerase poisons during vertebrate DNA replication using Xenopus egg extracts. These approaches allow for replication intermediates formed following poison treatment to be carefully monitored with high temporal resolution.