Synthetic biology is an engineering approach to biology. A synthetic biologist wants to describe biological molecules and their subdomains as well-defined parts of a molecular machine. To achieve this goal, synthetic biologists rebuild minimal functional biological systems from well-defined parts or they design new molecules that do not exist in nature but have new and useful functions. In short, these engineering approaches can be summarized as “rebuild, alter, and understand.” The Drosophila S2 Schneider cell is a useful tool for both rebuilding and redesigning approaches. S2 cells are phagocytic cells that easily take up large amounts of DNA from the cell culture. They, thus, have a high cotransfection rate, allowing the coexpression of up to 12 different proteins. We have developed a transient transfection protocol allowing the rapid and parallel analysis of wild-type and altered forms of a biological system. This chapter describes our methods to rebuild and better understand mammalian signaling systems in the evolutionary distant environment of Drosophila S2 cells.