Aims: This study aims to develop an efficient, and inexpensive transfection procedure using low cost polyethylenimine (PEI) as transfection reagent for overexpression of CYP2C9 in WRL 68 host cells. Introduction: Overexpression of CYP450 isozymes such as CYP2C9 has been a reliable approach in developing a high specificity in vitro tool for inhibition study of this liver enzyme. Transient or stable transfection are used to insert plasmid DNA into a mammalian host cell in order to produce proteins. There are many efficient transfection reagents available in the market for this purpose, however, it can be expensive, and a low-cost alternative is favorable. Methodology: Cytotoxicity of PEI was screened on four cell lines namely WRL 68, HepG2, MCF-7 and A549 using MTS assay. WRL 68 was transiently transfected with CYP2C9 plasmid using PEI and DNA concentration as well as transfection time was optimized for the best efficiency and expression. Expression of CYP2C9 protein was measured using qPCR and further evaluated with western blot analysis. Results: IC50 values of PEI are 0.327 ± 0.013 mg/mL (WRL-68), 0.395 ± 0.037 mg/mL (HepG2), 1.159 ± 0.032 mg/mL (A549) and 1.281 ± 0.000 mg/mL (MCF-7). Combination of 3ug plasmid DNA with 2.8 μM of PEI and 10.5 mM NaCl resulted in the highest transfection efficiency and expression after 48 hours. Conclusion: A low-cost and efficient PEI transient transfection procedure was optimized for CYP2C9 overexpression and useful for the purpose of further development of cell-based enzyme inhibition model.