AbstractThe ability to readily be designed, manufactured, and described has made cationic lipids one of the highly adaptable vehicles for the transfer of DNA, RNA, and numerous additional therapeutic compounds. In this article, we synthesized a variety of Tris‐(triazolyl)‐s‐triazine linker‐based cationic lipids with different types of anchoring groups, and assessed the transfection efficacy. Specifically, two identical tails like either two alpha‐tocopherol domains or cholesterol domains or aliphatic chains (saturated), and on the other hand, two different tails, alpha‐tocopherol domain and cholesterol domain or alpha‐tocopherol domain and aliphatic chain (saturated) were employed as hydrophobic motifs to synthesize the cationic lipids. The stability, size, and surface charge of liposomes were performed by dynamic light scattering technique. Cytotoxicity study on cell survival showed that the synthesized lipids are less hazardous to the cells despite the fact that cationic lipids have large molecular structure. The lipid having di alpha‐tocopherol domains demonstrated phenomenal β‐galactosidase protein expression and enhanced green fluorescence expression in the hepatocellular carcinoma cell line. Findings from the physico‐chemical and biological evaluations the developed lipids are compatible in various forms while maintaining low cytotoxicity and efficient carriers for nucleic acid delivery.