Transmembrane proton gradient plays a fundamental role in protein translocation across cellular membranes, including the transport of secreted enzymes from bacterial pathogens into host cells. Much attention has been devoted to understanding the machinery of such delivery and how it functions. Over the past decade, translocation of anthrax toxin has been widely studied not only because of its central role in the deadly pathogenesis of Bacillus anthracis, but also because that it is one of the most tractable toxins and thus serves as an attractive model for studying the translocation machinery that is dependent on proton gradient across membrane. Electrophysiological system for studying anthrax toxin translocation on planar lipid bilayers has yielded invaluable data on the process. However, the limitations of planar bilayers demand development of other sophisticated platforms to elucidate the mechanisms. This thesis will describe how droplet-interface bilayer (DIB) techniques, which have been developed as a model membrane system with distinct advantages over other platforms, are employed to study proton-gradient driven translocation of anthrax toxin. Our development of a novel perfusion system based on DIB allowed the proton gradient to be precisely and repeatedly switched over time. This system not only allows extensive studies in anthrax translocation process but is also broadly applicable to a variety of studies in membrane pores. Using this method, we discovered strong evidence for the current anthrax toxin translocation model, the conformational change of the toxin accompanied with its pH environments, and the small molecules that have interactions with the toxin.