Hepatitis A virus (HAV) is one of the most common causes of acute viral hepatitis worldwide. Formaldehyde is the currently used inactivating agent in HAV vaccine processing despite of its adverse effects. The current study aimed to evaluate both L-amino acid oxidase (LAO) and L-ascorbic acid (LAA) as alternative inactivants for HAV and the immunogenicity of inactivated HAV in mice. Vero cell line was used for cultivation of HAV. The cytotoxicity of LAO and LAA on Vero cells was evaluated using 3-(4,5-dimethylthiazol-2-yl) 2,5 diphenyl tetrazolium bromide (MTT) assay. The immunogenicity of each LAO- and LAA-inactivated HAV was examined in parallel with reference HAV vaccine in mice. Humoral (total IgG) and cellular immune responses (IFN-γ and IL-5) were evaluated in mice sera using ELISA. Both LAO and LAA could efficiently inactivate HAV within 30 and 36 hrs post treatment, respectively, at concentrations of 0.4 µgm/ml of LAO and 1.5 mg/ml of LAA. Inactivated vaccines were immunogenic to mice on both the humoral and cellular levels. LAO prepared vaccines showed a more promising immune reactivity than LAA prepared ones and alum-adsorbed vaccines were more immunogenic than non-adjuvanted ones. In conclusion, data recorded suggest that both LAO and LAA can be used as inactivating agents for HAV grown in cell culture. LAA- and LAO-inactivated HAV can be potential vaccines as they provide effective humoral and cellular immune responses comparable to that of the reference vaccine. The stability of test vaccines is recommended to be traced at different thermal conditions, in addition to different stabilizers and different pharmaceutical formulations must be tested trying to produce a lyophilized formula for long-term stability.