The continuing rise in cancer morbidity and mortality all over the world makes cancer the No. 2 cause of death in humans, posing a major public health challenge. Currently, surgery, radiotherapy, and chemotherapy are the three major approaches to treat cancer. However, the disadvantages of serious side effects, and poor therapeutic effect of the aforementioned methods in many clinical cases have become a burning question to be solved. DNA topoisomerases (Topos) are the key enzymes for DNA replication, transcription, recombination and repair, and have been regarded as target enzymes for some anticancer drugs, leading to the development of some successful anticancer drugs now in clinical use. Moreover, medicinal plants with numerous bioactive compounds are one of the most important sources for the discovery and development of anticancer drugs. In an active effort to search for Topo inhibitors from medicinal plants, affinity selection-ultrafiltration/high performance liquid chromatography-mass spectrometry (AS/UF-LC-MS) is among the most efficient techniques in term of sensitivity, throughput, and wide applicability. This review summarized the most recent advances in screening Topo inhibitors with AS/UF-LC-MS from medicinal plants over the past 15 years, with focus on structures, functions, the mechanisms of action of Topos and the procedures of the AS/UF-LC-MS technology, with an aim to provide comprehensive insights for new drug research and the discovery of more effective leading compounds against Topos.