Glutathione exists in biological samples in the reduced form (GSH), as its disulfide (GSSG) and as a mixed disulfide (GSSR) with thiols (RSH). GSH is the most abundant low-molecular-mass thiol and plays important roles as a cofactor and as a main constituent of the intracellular redox status. Due to its own sulfhydryl (SH) group, GSH reacts readily with o-phthaldialdehyde (OPA) to form a highly stable and fluorescent isoindole derivative (GSH-OPA), which allows for sensitive and specific quantitative determination of GSH in biological systems by HPLC with fluorescence (FL) detection. In the present article we report on the utility of the novel, strongly disulfide bond-reducing thiol N-acetyl-cysteine ethyl ester (NACET) for the specific quantitative analysis of GSH and GSSG in the cytosol of red blood cells (RBC) as GSH-OPA derivative with FL (excitation/emission 338/458 nm) or UV absorbance (338 nm) detection. Unlike in aqueous solution, the derivatization of GSH in RBC cytosol yielded two closely related derivatives in the absence of NACET and only the GSH-OPA derivative in the presence of NACET. The HPLC method was optimized and validated for human RBC and applied to measure GSH and GSSG in RBC of healthy subjects. Basal GSH and GSSG concentrations were determined to be 2340 ± 350 μM and 11.4 ± 3.2 μM, respectively, in RBC of 12 healthy young volunteers (aged 23–38 years). The method was also applied to study the effects of nitrite on the glutathione status in intact and lysed human RBC. Nitrite at mM-concentrations caused instantaneous and considerable GSSG formation in lysed but much less pronounced in intact RBC. GSH externally added to lysed RBC inhibited nitrite-induced methemoglobin formation. Our findings suggest that nitric oxide/nitrite-related consumption rate of GSH, and presumably that of NADH and NADPH, could be of the order of 600 μmol/day in RBC of healthy subjects.