On a commercial scale, the vaccine against yellow fever is based on replication of the attenuated virus in embryonated eggs [2]. A very promising alternative platform to the production of the virus in eggs, which has spread as a worldwide trend, is production using mammalian cell lines. The advantage of using established animal cell lines in vaccine production lies in their greater reproducibility and convenience when compared to primary cells and animal models, as well as their great potential for large-scale production. Such cell lines can be cultivated in bioreactors, under monitored and controlled conditions, providing suitable conditions for viral replication. In this protocol, we describe the production of 17DD yellow fever virus from the cultivation of cell line using agitated bioreactors.