Methods for in vivo monitoring of redox changes in different cellular compartments have been developed in recent years, and are mostly based on redox-sensitive variants of the green fluorescent protein (GFP). However, due to the thermodynamic stability of the introduced reactive disulfide bond, these sensors are limited to reducing compartments such as the cytosol and the mitochondria, and are not suited for more oxidizing environments such as the endoplasmic reticulum (ER). To overcome this problem, a family of redox-sensitive GFP variants that differed in their midpoint potential has been developed by the group of Remington (University of Oregon) and tested in vitro. Here, we report the first in vivo use of these novel roGFP1 variants for the measurement of redox conditions within the ER and cytosol in the yeast Pichia pastoris. With the fluorescence data obtained, it was possible to determine the reduction potential of the two compartments. Thereby, we could show that the ER, which is required for oxidative protein folding, is indeed more oxidizing than the cytosol. Contrary to previous results with roGFP, the optimized roGFP1_iE and roGFP1_iL constructs were not completely oxidized, and are therefore useful sensors for monitoring the ER under conditions when it is even more oxidized.