The threading of proteins through narrow channels within a cell is a crucial biological process, which is essential for cellular protein trafficking and protein degradation. This translocation process is also used by pathogens such as anthrax and C. Difficile, which inject their lethal toxins through the cell membrane in order to hijack vital cell machinery. The motor proteins driving this translocation process are called protein translocases. Despite the ubiquity of protein translocation in biology, the underlying mechanisms remain poorly understood. Improving our knowledge of translocation will have implications on our understanding of severe pathologies, which have been linked to the dysfunction of translocases (e.g., cancers, Alzheimer’s).I intend to design novel biorelevant in vitro tools to investigate the inner workings of membrane-associated protein translocases. The translocase will be reconstituted in a cell-like microenvironment, while allowing real time control over biologically appropriate external conditions that may affect its function in vivo (e.g., voltage/pH gradient across membrane). Our innovative approach is based on the integration of novel lipid systems into a combination of optical tweezers and electrophysiology techniques. The first translocase targeted is FtsH, whose role is to dislocate proteins from membranes. We will directly measure the translocation velocity and step size of this membrane-associated translocase with unprecedented detail, while being able to control the protonmotive force.These new assays will advance considerably single-molecule investigations of membrane proteins, which represent 30% of all proteins and are prime drug targets. This work will lay foundations towards elucidating the mechanisms behind other crucial membrane translocases, in particular transmembrane protein transporters.