Transgenic cell lines that produce biopharmaceutical proteins are widely utilized in the biotech industry and the demand is not predicted to decline in the near future. For obtaining an industrially usable cell line, process components like expression vector, host cell line and transfection method need to be carefully selected. Due to all practical reasons, the industry prefers to use the most conventional Chinese hamster ovary (CHO) cell line. The generation of recombinant cell lines is known to be time-consuming, labor-intensive and expensive. Therefore, the several steps of this process are under constant development. One of the first work packages is transfection, where genes encoding for the therapeutic protein are taken into mammalian host cells. In this study, we aimed to generate a more cost-effective transfection procedure using the electroporation based technology of nucleofection. This method is favored by the researchers for its high and reproducible transfection efficiency, but also known for the high cost and lack of public information on its components and related consumables. As a result of this study, a novel nucleofection solution was developed for the transfection of CHO-DG44 cells, showing comparable if not better performance over the commercial Lonza's solution in terms of transient and stable expression of recombinant proteins. The transfection was further improved by selecting a more effective nucleofection program and by linearizing the plasmid prior to transfection. These enhancements, optimized on the basis of the biotherapeutic protein production, are potentially advantageous for any research requiring a large number of efficient transfection experiments.