Disulfide bonds drive protein correct folding, prevent protein aggregation, and stabilize three-dimensional structures of proteins and their assemblies. Dysregulation of this activity leads to several disorders, including cancer, neurodegeneration, and thrombosis. A family of 20+enzymes, called thiol-isomerases (TIs), oversee this process in the endoplasmic reticulum of human cells to ensure efficacy and accuracy. While the biophysical and biochemical properties of cysteine residues are well-defined, our structural knowledge of how TIs select, interact and process their substrates remains poorly understood. How TIs structurally and functionally respond to changes in redox environment and other post-translational modifications remain unclear, too. We recently developed a workflow for site-specific incorporation of non-canonical amino acids into protein disulfide isomerase (PDI), the prototypical member of TIs. Combined with click chemistry, this strategy enabled us to perform single-molecule biophysical studies of PDI under various solution conditions. This paper details protocols and discusses challenges in performing these experiments. We expect this approach, combined with other emerging technologies in single-molecule biophysics and structural biology, to facilitate the exploration of the mechanisms by which TIs carry out their fascinating but poorly understood roles in humans, especially in the context of thrombosis.