BACKGROUND L-carnitine (LC) is commonly used in the treatment of valproate-induced hyperammonemia (VIHA). LC prevents the production of ammonia with no significant effect on renal ammonia excretion. This study was conducted to evaluate the effect of sodium benzoate (SB) and phenyl acetate (PA) on reducing VIHA. METHODS Eight groups treated with Sodium Valproate (SV) at 300 mg/kg and 15 minutes later with normal saline, SB (144 mg/kg), PA (0.3 g/kg), LC (2.5 g/kg), SB (144 mg/kg) plus PA (0.3 g/kg), or SB (144 mg/kg) plus PA (0.3 g/kg) plus LC (2.5 g/kg), intraperitoneally. Other groups were exposed to normal saline, SB, LC or PA alone. Animal's motor function and serum ammonia, lactate, and sodium levels were assessed at 0.5, 1, and 1.5 hours after the SV injection. RESULTS The results showed that LC reduced SV-induced hyperammonemia just at one and half-hour after treatment (P<0.001). PA, alone or in combination with other antidotes, reduced serum ammonia at all evaluated times (P<0.001). LC improved the impaired motor function of animals only at 1.5 hours, while PA, alone or in combination decreased the motor function scores at different times. However, SB administration alone did not change SV-induced hyperammonemia or motor function impairment. There was no significant difference in the level of serum aminotransferases, blood urea nitrogen, and creatinine between groups. CONCLUSIONS These findings define that PA had a better therapeutic effect on valproate-induced hyperammonemia in comparison with SB. Co-administration of LC with PA ameliorated the elevated levels of ammonia and may relieve potential therapeutic application against acute SV intoxication.