To understand how intracellular proteins respond to oxidative stresses, the redox status of the target protein, as well as the intracellular redox potential (E-GSH), which is defined by the concentrations of reduced and oxidized glutathione, should be observed simultaneously within living cells. In this study, we developed a method that can monitor the redox status of thioredoxin (Trx) and E-GSH by direct NMR observation of Trx and glutathione within living cells. Unlike the midpoint potential of Trx measured in vitro (similar to -300 mV), the intracellular Trx exhibited the redox transition at EGSH between -250 and -200 mV, the range known to trigger the oxidative stress-mediated signalings. Furthermore, we quantified the contribution of Trx reductase to the redox status of Trx, demonstrating that the redox profile of Trx is determined by the interplay between the elevation of EGSH and the reduction by Trx reductase and other endogenous molecules.