This study was conducted to determine the effect of pre-harvest glyphosate application on spring wheat quality characteristics, ranging from kernel quality to baking quality. Two wheat cultivars were grown in three locations, and glyphosate was applied at the recommended rate at the soft dough stage (early application) and the ripe stage (recommended application time). When glyphosate was applied at the soft dough stage, kernel weight, wheat protein and wet gluten decreased significantly (p ≤ 0.05), however, gluten index significantly increased (p ≤ 0.05). Dough quality, farinograph stability, and quality number were significantly (p ≤ 0.05) higher when glyphosate was applied at the soft dough stage, and absorption and the mixing tolerance index were lower. As for baking quality, loaf volume and mix time were significantly (p ≤ 0.05) higher in the treated samples in comparison to the untreated control, and other baking quality characteristics did not show any significant difference. Overall, the results indicate that spring wheat quality characteristics are impacted to the greatest degree when glyphosate is applied earlier than recommended at the soft dough stage, as opposed to the recommended application at the ripe stage of physiological development.