Pep-1 is a promising peptide tool that delivers proteins and peptides into cells with conserved bioactivity. Pep-1 has great potential because of the high efficiency and lack of toxicity. The mechanism of Pep-1-mediated transport is not fully understood. In my thesis, droplet-interface bilayer (DIB) has been used for the mechanistic studies of Pep-1. Here, DIB is developed for different functions such as quantitation of protein translocation, solution exchange to a formed bilayer and simultaneous observation of multiple membranes. Research work on Pep-1 with DIB reveals that the negative charge of the inner membrane leaflet plays a significant role in promoting cargo translocation. Further investigation indicates that the transport efficiency is time-dependent and complex formation of peptide and cargo is required.