Transient gene expression, an attractive technology that enables the rapid production of recombinant proteins for the development of biopharmaceutical candidates at an early stage, offers an interesting alternative to the time-consuming generation of stable recombinant cell lines. In this study, a highly efficient transient gene expression system was established by optimizing significant factors; namely, the host cells, lipid-based transfection reagent, enhancer, and expression vector. HEK293E and Expi293 F cells were evaluated to determine the best host cells to use, and the cell passage number and initial cell density were optimized for high transient gene expression. Cationic liposomes were selected as the vehicle for plasmid DNA delivery and optimized with respect to the co-lipid complex and DNA-to-lipid ratio. Among the cationic lipids, DC-Chol/DOPE gave a more than 2-fold increase in production yield. Following gene delivery into cells, butyric acid (a histone deacetylase inhibitor) supplementation increased protein production by a maximum of 4-folds through transcriptional regulation. Additionally, an expression vector bearing the E77 element at the 3' end of the polyadenylation signal sequence further enhanced the human-Fc and bevacizumab yields by 7% and 15%, respectively. Finally, our novel combination method was successfully applied to human IgG and Fc protein production using Expi293?F cells.