The Hsp70 chaperone system performs a critical function in the maintenance of protein homeostasis and cellular health. One such role is the folding of nascent proteins from a polypeptide chain at synthesis or the refolding of proteins that have engaged in incorrect folding pathways. This refolding action is thought to occur by remodelling of the client protein by the chaperone machinery; however, many of the specific molecular interactions that underpin this process have yet to be well characterised. This study aims to interrogate these processes through the use of single-molecule Fluorescence Resonance Energy Transfer (smFRET) within a Total Internal Reflection Fluorescence (TIRF) framework. Double-cysteine mutants of firefly luciferase (Fluc) that could be site-specifically labelled with fluorophores were generated to act as protein-folding sensors to be refolded by the chaperone machinery. In concert with enzymatic luminescence assays, smFRET experiments are to be performed to monitor conformational changes experienced by these clients as they interact with the human Hsp70 system (HspA8, DnaJA2, Hsp110). We aim to resolve mechanistic details regarding the number of cycles of Hsp70 binding and release that are required for productive refolding, as well as identify key folding intermediates involved in this process. Additionally, by using folding-sensors with distinct labelling positions, we hope to map out how different regions within the protein structure fold and interact with molecular chaperones.