This study introduces redox-sensitive green fluorescent protein (roGFP) indicators to monitor redox changes in living cells. By introducing disulfide bonds near the chromophore of Aequorea victoria green fluorescent protein (GFP), roGFPs exhibit reversible changes in fluorescence excitation maxima upon oxidation-reduction transitions. Crystal structure analysis of roGFP2 reveals a highly strained disulfide bond and localized structural changes upon oxidation, which likely account for the observed spectral shifts. roGFP1, targeted to mitochondria in HeLa cells, responds to membrane-permeable reductants and oxidants, indicating that the mitochondrial matrix is highly reducing with a midpoint potential near −360 mV. These roGFPs provide a noninvasive method for studying redox processes in living cells, offering insights into cellular redox homeostasis and its role in various cellular functions.This study introduces redox-sensitive green fluorescent protein (roGFP) indicators to monitor redox changes in living cells. By introducing disulfide bonds near the chromophore of Aequorea victoria green fluorescent protein (GFP), roGFPs exhibit reversible changes in fluorescence excitation maxima upon oxidation-reduction transitions. Crystal structure analysis of roGFP2 reveals a highly strained disulfide bond and localized structural changes upon oxidation, which likely account for the observed spectral shifts. roGFP1, targeted to mitochondria in HeLa cells, responds to membrane-permeable reductants and oxidants, indicating that the mitochondrial matrix is highly reducing with a midpoint potential near −360 mV. These roGFPs provide a noninvasive method for studying redox processes in living cells, offering insights into cellular redox homeostasis and its role in various cellular functions.