This research assessed the effect of varying inclusions of monosodium glutamate (MSG) on serum electrolytes and antioxidative enzymes of laying hens. A total of 300 Isa Brown point-of-lay pullets, aged 16 weeks, were evenly allocated to six distinct experimental lots, each containing different levels of MSG (0.00, 0.25, 0.50, 0.75, 1.00 and 1.25 g/kg). Throughout the study, the experimental hens were granted unrestricted freedom to diets, and clean water was consistently provided. In the twelfth week, five hens per replicate were chosen randomly after an overnight fast for blood collection through the wing veins. Standard procedures were employed to analyze serum electrolytes, including Na+ (sodium), K+ (potassium), and Cl- (chloride), together with antioxidant enzymes such as T-OAC (total antioxidant capacity), SOD (superoxide dismutase), and GSH-Px (glutathione peroxidase). Additionally, an assessment of the oxidative stress indicator malondialdehyde (MDA) was undertaken. The findings revealed that the addition of MSG at 1.00 and 1.25 g/kg in the feed significantly increased the serum Na+ concentration of the pullets, while the serum K+ level decreased significantly at the 1.25 g/kg MSG administration level, compared to the hens in the control group. In contrast, the blood Cl- level significantly decreased with MSG inclusion at the 0.50 g/kg level. Moreover, MSG inclusion levels at or above 0.75 g/kg led to an increase in MDA concentration, accompanied by a significant reduction in serum SOD, GSH-Px, and T-AOC levels. Thus, including MSG at levels exceeding 0.50 g/kg in the diet may potentially predispose laying hens to oxidative stress and other physiological imbalances.