Improving the efficiency of the methods to estimate the sensitivity and specificity of vaccines is of great need and value. Immunoperoxidase and neutralization/RT-PCR were accomplished to estimate the efficiency of the human rotavirus VP8 structural protein candidate subunit vaccine previously examined using a neutralization test in another study of our group. This neutralization test was performed for expressed proteins of common P genotypes (P8, P4, and P6) of human rotaviruses either separately or as a mix. In our present study, results showed that 1.4, 1.6, and 2 log10 reduction of initial viral titre (105 infectious viral units) were achieved with 1/32 dilution of the rabbit anti-sera which produced against the tested P8 protein using immunoperoxidase, neutralization followed by RT-PCR, and neutralization followed by nested RT-PCR respectively. Also, 0.9 log10, 1.1 log10, and 1.5 log10 reductions of initial viral titre were achieved with 1/32 dilution of the rabbit anti-sera which produced against the tested P4 and P6 proteins using immunoperoxidase, neutralization followed by RT-PCR, and neutralization followed by nested RT-PCR respectively. The capacity of neutralization decreased with increasing the dilution of rabbit anti-sera. No significant difference was observed between the results of immunoperoxidase in relation to the neutralization test. Neutralization followed by RT-PCR for rotaviruses in MA104 cells infected with viruses showed higher sensitivity in the estimation of the human rotavirus Wa strain titre directly or after neutralization with antibodies of P8, P4, and P6 expressed proteins. Neutralization followed by nested RT-PCR may be recommended to estimate the efficiency of the human rotavirus VP8 candidate recombinant subunit vaccine.