An improved gas chromatographic method using flame ionization detection (GC-FID) for quantification of free glutathione (GSH), free glutathione disulphide (GSSG), total free glutathione (TFG) and protein bound glutathione (PSSG) in wheat flour has been developed. The process involves extraction of GSH and GSSG, wherein the extraction efficiencies of five different solvent systems, including acetonitrile, methanol, trichloroacetic acid (TCA), meta-phosphoric acid (MPA) and perchloric acid (PCA) were tested. A comparison between 1,4-dithiothreitol (DTT) and tris(2-carboxyethyl)phosphine (TCEP) was made in order to evaluate their efficacy to reduce GSSG to GSH. Results showed that TCEP reduced GSSG to GSH, and released GSH from PSSG, whereas DTT did not reduce GSSG to GSH at a sufficient level at the tested experimental conditions. The method showed a linear response for the concentration in the range of 6.25–50 nmol/mL. The limit of detection and the limit of quantification values were 2.03 and 6.08 nmol/mL, respectively. The level of GSH in 40 different wheat flours was in the range of 19.3–137 nmol GSH/g, while GSSG and PSSG levels ranged from 4.32 to 200 and 5.15–61.4 nmol GSH equivalents/g flour. Pearson's correlation coefficients showed minor positive correlations for baking volume, protein and wet gluten to GSH.