Gene therapy bears great potential for the cure of a multitude of human diseases. Research efforts focussed on the use of viral delivery vectors in the past decades, neglecting non-viral gene therapies of physical or chemical origin due to low transfection efficiency. However, side effects such as activation of oncogenes and inflammatory reactions upon immune cell activation are major obstacles impeding the clinical applicability of viral gene therapy vectors. The aim of this study was the development of a non-viral gene delivery system based on plasmid-loaded human serum albumin nanoparticles, which are biocompatible, biodegradable, and non-toxic in relevant concentrations. The surface of said nanoparticles was modified with different cell penetrating peptides, namely Tat, nona-arginine R9, and the penetratin analogue EB1. We hypothesise that the surface modified nanoparticles can effectively enter HEK 293T cells based on the cell penetrating properties of the different peptides attached. A variety of inhibitors were used targeting distinct uptake pathways in an effort to understand the mechanisms utilized by the various cell penetrating peptides on the surface of the nanoparticles. A significant increase in transfection efficiency compared to free DNA or polyplexes was seen for these novel delivery vectors.