Rabies is a zoonotic disease that can be prevented by vaccination. The confirmation of rabies virus inactivation is a critical step during the vaccine quality test; however, the current protocol conducted in Japan requires a large number of mice. The development and introduction of animal-free alternative assays are essential from the perspective of the 3Rs (reduction, refinement, and replacement) of animal testing. Here, we propose a novel inactivation assay for confirming the complete inactivation of the viable rabies virus using cultured Neuro-2a cells and an enzyme-linked immunosorbent assay (ELISA). The detection ability of ELISA was similar to that of a direct immunofluorescence assay, with the detection limit of ELISA being as low as 0.014 focus forming units/test. These results suggest that the assay could be used as a viral inactivation test. In comparison with a traditional in vivo assay, this assay has a higher detection ability, an objective interpretation, and would shorten the test duration from 25 days to 8 days.