Insect Drosophila melanogaster S2 cell was developed as plasmid-based and, therefore, a nonlytic expression system for functional foreign proteins. To achieve multiple protein expressions, it was suggested that baculovirus be used on S2 cell system because baculovirus can infect S2 cells but cannot replicate inside the cells. Therefore, establishment of baculovirus infection conditions is the first important step and this should be properly optimized for production yield. We used statistical methodology to optimize the baculovirus infection conditions using green fluorescent protein (GFP) as a reporter protein. Consequently, we arrived at optimal infection conditions through a statistical regression method. The secreted GFP yield from vMT-GFP baculovirus-infected wild-type S2 cells under optimal infection conditions was > 15-fold higher than that under nonoptimal conditions and comparable to that from stably transfected recombinant S2 cells.