Barley is a staple crop of major global importance and relatively resilient to a wide range of stress factors in the field. Transgenic reporter lines to investigate physiological parameters during stress treatments remain scarce. We generated and characterized stable homozygous barley lines (cv. Golden Promise Fast) expressing the genetically-encoded biosensor Grx1-roGFP2, which indicates the redox potential of the major antioxidant glutathione in the cytosol. Our results demonstrate functionality of the sensor in living barley plants. We determined the glutathione redox potential (EGSH) of the cytosol to be in the range of 308 to -320 mV. EGSH was robust against a combined NaCl (150 mM) and water deficit treatment (-0.8 MPa) that caused growth retardation and showed only a minor oxidation after 96 h of treatment. We conclude that the generated reporter lines are a novel resource to study stress resilience in barley.