Disulfide bond formation in vivo is linked to many essential intracellular processes; protein regulation and signaling, chemical transformations, and oxidative protein folding. Oxidative protein folding is an enzyme catalyzed process which is controlled by dedicated protein thiol oxidoreductases. In this work the oxidative protein folding within the mammalian endoplasmic reticulum (ER) is examined from an enzymological perspective. Evidence for the rapid reduction of PDI by reduced glutathione is presented in the context of PDI-first pathways. Next, strategies and challenges for the determination of the concentrations of reduced and oxidized glutathione and of the ratios of PDIred:PDIox is discussed. After a discussion of the use of natively encoded fluorescent probes to report the glutathione redox poise of the ER, a complementary strategy to discontinuously survey the redox state of as many redox-active disulfides as can be identified by ratiometric LC–MS–MS methods in order to better understand redox linked species. Next, we investigate the specificity of the human Mia40/lfALR system towards non-cognate unfolded protein substrates to assess whether the efficient introduction of disulfides requires a particular amino acid sequence context or the presence of an IMS targeting signal. Mia40 is found to be effective oxidant of non-cognate substrates, but is an ineffective protein disulfide isomerase when its ability to restore enzymatic activity from scrambled RNase is compared to that of protein disulfide isomerase. Mia40’s ability to bind amphipathic peptides tested by the insulin reductase assay. The consequences of these studies, mitochondrial oxidative protein folding, and the transit of polypeptides is discussed. Finally, the development of disulfide linked genetically encoded fluorescent probes for analyte-specific imaging are demonstrated. Current classes of intracellular probes depend on the selection of binding domains that either undergo conformational changes on analyte binding or can be linked to thiol redox chemistry. Here, novel probes were designed by fusing a flavoenzyme, whose fluorescence is quenched on reduction by the analyte of interest, with a GFP domain to allow for rapid and specific ratiometric sensing. Two flavoproteins, Escherichia coli thioredoxin reductase and Saccharomyces cerevisiae lipoamide hydrogenase, were successfully developed into thioredoxin and NAD+/NADH specific probes respectively and their performance was evaluated in vitro and in vivo. These genetically encoded fluorescent constructs represent a modular approach to intracellular probe design that should extend the range of metabolites that can be quantitated in living cells.