In vaccine and viral vector production, impurity clearance is an essential purification step and required for drug approval as residual impurities may impact patient safety. Additionally, impurities such as extracellular nucleic acids present in process intermediates can interfere with and reduce the efficiency of various purification steps. A critical step in the purification process is reduction in the size and quantity of extracellular nucleic acids by endonuclease enzymes. Benzonase® endonuclease is a genetically engineered nuclease from Serratia marcescens, able to attack and degrade all forms of DNA and RNA. Because of its essential role in vaccine and viral vector processes, it is manufactured under GMP according to ICH Q7, and has a Drug Master File in place with the United States Food and Drug Administration (FDA) which can be cited in regulatory filings. Both the World Health Organization (WHO) and the FDA recommend clearance of DNA to a level of less than 10 ng/dose and fragments less than 200 base pairs in length. This application note summarizes the benefits of incorporating Benzonase® endonuclease in a polio virus type 3 production process.