Background: The most important antioxidant for erythrocytes is glutathione. In this study, a non-enzymatic recycling spectrophotometric method was developed to measure oxidized and reduced glutathione (GSH) levels in erythrocytes. The newly developed method consists of two steps. In the first step, the levels of GSH were determined in the sample. Subsequently, total glutathione levels were measured by reducing the oxidized glutathione (GSSG) with sodium borohydride (NaBH₄). Half of the difference between total glutathione and GSH gives the GSSG.Results: The new method was linear between 0 and 3000 μmol/L (r² = 0.999). The percentage recovery measured by the developed method was 100.2 ± 2.4%. The total precision of the total glutathione, GSH and GSSG was 1.26%, 1.02% and 6.65%, respectively. GSH levels were significantly lower in patients with type 2 diabetes mellitus (DM), while GSSG levels were significantly higher (p < 0.05). Hemoglobin A_1c(HbA_1c) levels were negatively correlated with GSH levels, whereas positively correlated with GSSG (p < 0.05).Conclusions: In conclusion, this method is easy to apply in routine practice with high accuracy, precision and reproducibility.