In this study, dieosin glutathione disulfide (Di-E-GSSG) was synthesized by the reaction of eosin isothiocyanate with GSSG. Di-E-GSSG had low fluorescence which increased ∼70-fold on reduction of its disulfide bond. The substrate was used to monitor the disulfide reductase activity of PDI. Di-E-GSSG is the most sensitive pseudo substrate for PDI reductase activity reported to date. This probe was further used as an analytical reagent to develop an end point assay for measuring the redox state of PDI. The reduction of Di-E-GSSG by reduced enzyme was studied in the absence of reducing agents and the redox state of PDI was monitored as a function of the stoichiometric changes in the amount of eosin-glutathione (EGSH) generated by the active-site dithiols of PDI. The redox state of PDI was also studied under variable [GSH]/[GSSG] ratios. The results indicate that PDI is in ∼1/2-reduced state where the [GSH]/[GSSG] ratio is between 1:1 and 3:1, conditions similar to the lumen of endoplasmic reticulum or in the extracellular environment. On the other hand, [GSH]/[GSSG] ratios of ≥8:1, such as in cytosol, all active-site thiols would be reduced. The study was extended to utilize Di-E-GSSG to investigate the effect of variable redox ratios on the platelet surface PDI reductase activity.