This chapter provides an overview of the biological, chemical, and physical factors involved in enhancing the ability of in vitro systems to produce baculoviruses for insect pest control purposes. Baculoviruses have been isolated from a wide variety of pest insects from several orders, including Lepidoptera and Coleoptera. Baculoviruses have circular, covalently closed double-stranded DNA and are members of the Baculoviridae family. The Nudibaculovirinae consist of the nonoccluded baculoviruses, which are singly enveloped and do not produce occlusion bodies (OB). Numerous biological factors affect the level of in vitro production and virulence of baculovirus extracellular viruses and OB. Cell lines from the same species but different tissues vary in their ability to produce baculoviruses as well. The quantity and quality of baculovirus production in cell culture is also affected by the strain of the virus and the type of infection process it undergoes.