Cell transfection efficiency often determines the success of cell-based gene therapy. Cell transfection via Nucleofector technology yields high transfection efficiency and low cytotoxicity. However, owing to trade secrecy, the components in each buffer are unknown, which not only increases the cost of electroporation studies but also limits the application of Nucleofector in clinical cell-based gene therapies. Thus, we developed a three-step method to determine the optimal conditions, including buffer, program, and additional polymer, in electroporation for multiple cancers and stem cell lines. This method could reduce the cost, allow researchers to find the optimal electroporation conditions for their cell lines of interest, and greatly boost the application potential of electroporation in clinical cell-based gene therapies.