Gene transfer and gene therapy studies require high-efficiency gene delivery reagents. By transferring the piece of DNA that we are interested in, we can alter the expression of certain gene or genes to further characterize its role in the cell function or in the organism's development, metabolism, immune system, etc. Transfection reagents that enable efficient delivery of the DNA to the cells are important tools in the molecular and cellular biology studies. There are chemical products and tools that have been used for transfection of the cells but they are not as efficient as desired or they can induce cytotoxicity. It is crucial to design and generate new transfection reagents to further support the field of biotechnology, molecular studies, cellular biology, and in vitro studies relying on them. The more efficient and the less cytotoxic compounds will be especially useful for the field. We synthesized a new set of benzimidazole-based transfection reagents that have higher efficiency to carry GFP expressing plasmid in to the mammalian cells compared with the commercially available ones with low cytotoxicity. GFP expression levels were tracked by flow cytometry to determine the transfection efficiencies. Benzimidazole-based transfection reagents can be safely used for transfection studies in tissue culture as well as in gene therapy applications due to their high efficiency in the gene transfer to the mammalian cells.