The maintenance and regulation of protein homeostasis is heavily dependent on a vast network of molecular chaperones, which together act to prevent the formation and accumulation of misfolded and aggregated forms of proteins. Interactions between chaperone proteins and their clients are often transient and heterogeneous in nature, which make them difficult to study using traditional bulk-phase measurements due to ensemble averaging. Recently, single-molecule techniques have emerged as a powerful tool to study chaperone function since individual proteins can be monitored in real time, enabling the characterization of rare and transient species that may be present. Consequently, we aimed to develop a protein folding-sensor based on the chloride intracellular channel (CLIC) and rhodanese proteins that can be used in single-molecule experiments to report on folding transitions upon interaction with chaperones. By utilizing the ability of fluorophores to be quenched when in close proximity to certain amino acids (e.g. tryptophan) or identical fluorophores, studies were conducted to attempt to develop CLIC1 into a folding-sensor that could report on conformational transitions via changes in fluorescence intensity. Single-molecule imaging of various site-specifically labelled CLIC1 mutants revealed that CLIC1 undergoes significant conformational dynamics in solution; however, no significant difference in CLIC1 conformational dynamics was observed when incubated under non-denaturing versus denaturing conditions. Rhodanese was developed into a folding-sensor by introducing two fluorophore pairs that could be used to report on folding transitions via changes in FRET. Ensemble-based fluorometer assays revealed that unfolding of rhodanese by chemical denaturants could be monitored via a significant changed in FRET efficiency. Furthermore, single-molecule analysis revealed that individual rhodanese molecules exhibited high FRET efficiencies (~80%) when correctly folded, which is in agreement with previous studies [1]. Collectively, these results suggest that rhodanese, but not CLIC1, could be implemented as a folding-sensor in single-molecule experiments to study chaperone function.