This study developed a novel approach for evaluating the infectivity of enteric viruses without cell culture. Cumulative carbonyl groups on the viral capsid protein were labeled using biotin hydrazide, and the biotinylated virions were separated using a spin column filled with avidin-immobilized gel. Rotavirus was treated with free chlorine at an initial concentration of 0.3 mg/L for 3 min, and the log reduction in the infectious titer was 0.19 log (standard deviation, SD = 0.05). The log reduction of rotavirus treated with free chlorine at an initial concentration of 0.6 mg/L for 3 min was 2.6 log (SD = 0.37). No significant reductions in the amplicon copy numbers were observed in these free chlorine-treated samples. The recovery levels of intact virions in the first three fractions after biotin-avidin affinity chromatography were 76, 21, and 2.8%, while those of virions treated with free chlorine at an initial concentration of 0.3 mg/L for 3 min were 70, 23, and 5.6%. These results showed that the proposed approach could discriminate a 0.19 log infectivity-reduced population from an intact population, although no reduction in the amplicon copy number was observed. This novel method could be applied to noncultivatable enteric viruses such as human norovirus and sapovirus, and it could be very helpful for evaluating the viral inactivation efficiencies of intervention measures.