For targeted cellular therapy using both synthetic and natural biomaterials, there is a need for better understanding of cell penetration mechanisms. Recently, nanoparticles (NPs) coated with mixed-ligand self-assembled monolayers exhibiting order in their molecular arrangement have been discovered to fuse with and penetrate through cell membranes in a passive, energy independent manner. The surface of these NPs contains amphiphilic molecules that are similar in structure to the surface of cell-penetrating peptides (CPPs), small endogenous biomolecules that facilitate cellular uptake by both passive and active membrane processes. CPPs have been identified to play a major role in viral transfection, and have been developed as potential nanocarriers for drug delivery. The similarities between CPPs and mixed-ligand NPs suggest that the driving force for their fusion with lipid bilayers can be explained as a general physical chemistry process. Presented within is an investigation into the synthesis and characterization of mixed-ligand amphiphilic NPs for the quantification and elucidation of their interaction with lipid bilayers. By careful systematic variation of NP properties, we have developed a theory for the mechanism of fusion. We hope that the elucidation of the driving forces forNP-lipid bilayer fusion enables the fundamental understanding ofmembrane dynamics with molecules such as CPPs, with the overall intention of understanding the more complex cellular membrane and its penetration mechanisms. A major implication of this work is that the knowledge of membrane-transport processes can be exploited to aid the design of more efficient therapeutic applications.