Sodium valproate (VPA) has 16 known metabolites in humans. The 2-ene-VPA has anti-convulsant efficacy and 4-ene-VPA is reported to contribute in VPA hepatotoxicity. The formation of 4-ene-VPA is catalyzed by cytochrome P450 2C9 (CYP2C9). CYP2C9 allele mutation is closely related to the attenuation of the enzymatic activity and 4-ene-VPA production. In the present work, VPA, 2-ene-VPA, and 4-ene-VPA in serum of patients receiving VPA were determined and the correlation between CYP2C9 polymorphism and 4-ene-VPA concentration was examined. Blood samplings in 68 patients were performed at two time-points (peak and trough) and one sample blood obtained from 50 healthy volunteers for genotype evaluation. Patients were divided into three groups (22 cases of monotherapy, 19 cases of enzyme inducer therapy, and 27 cases of polytherapy). There was a significant reduction in concentration of VPA and 4-ene-VPA between peak and trough time. In peak concentration, there was a significant correlation between 2-ene-VPA and VPA in all groups. The concentration of 4-ene-VPA in the enzyme inducer and polytherapy group was significantly higher than that of the monotherapy group. The allele frequencies of CYP2C9*1, CYP2C9*2, and CYP2C9*3 were 88.97%, 8.09%, and 2.94% in the patient group and 91%, 6%, and 3% in the normal group, respectively. There was no significant difference in allele frequency in two groups. Mutated alleles didn't have any significant effect on 4-ene-VPA production. No patient showed toxic level of 4-ene-VPA or saturation of ß-oxidation pathway. In conclusion, the role of CYP2C9*2 and CYP2C9*3 in attenuation of 4-ene-VPA formation cannot be confirmed.