The production of the nucleopolyhedrovirus (NPV) of the tobacco cutworm, Spodoptera litura was studied to determine the optimum conditions for in vivo production using homologous host larvae reared on artificial diet. We compared NPV production in 7, 8, 9 and 10-day-old larvae and found that 9-day-old larvae gave the highest virus yield. The optimum dose was determined by infecting 9-day old larvae (weighing 125–155 mg) with a range of doses. The maximum NPV production achieved was 3.91 × 109 polyhedral inclusion bodies (PIBs) per larva using an inoculum of 4.8 × 106 PIBs. A 7-day incubation period was found to be optimum for NPV production. Moribund larvae were found to be more suitable for handling and for the mass production of virus than dead ones.