The liposomal systems proved remarkably useful for the delivery of genetic materials but enhancing their efficacy remains a significant challenge. While structural alterations could result in the discovery of more effective transfecting lipids, improving the efficacy of widely used lipid carriers is also crucial in order to compete with viral vectors for gene delivery. Herein, we developed formulations of commercially available lipid, 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) with synthetic amino acid based cationic lipids. Two cationic lipids were synthesized using amino acids, with either cystine (CTT) or arginine (AT) in the head group. These lipids were used to formulate co-liposomal structures with different lipid compositions. The liposomal formulations were broadly categorised into two types: amino acid-based liposomes without DOTAP (CTTD and ATD) and those with DOTAP (DtATD and DtCTTD). Optimized lipid-DNA complexes of DOTAP-incorporated formulations (DtATD and DtCTTD) exhibited enhanced efficacy in transfection compared to formulations lacking DOTAP as well as commercial formulations such as DOTAP:DOPE. Notably, DtCTTD displayed superior transfection capabilities in prostate cancer (PC3) and lung cancer (A549) cell lines when compared to the widely used commercial transfection reagent, Lipofectamine. Collectively, the findings from this study suggest that DOTAP-incorporated formulations derived from amino acid-based liposomes, hold promise as effective tools for improving transfection efficacy with reduced toxicity, offering potential advancements in gene delivery applications.