Influenza is an infectious respiratory disease that has significant morbidity and a high mortality rate. The immune responses induced against both seasonal and potentially pandemic strains of the influenza virus are of fundamental importance. The microneutralization (MN) assay is an extremely important virological test for the evaluation of vaccines, and therefore of the antibody response. However, there is not always a correspondence between the results obtained from the same samples, owing to the difficult readout of the cytopathic effect (CPE) in the case of live viruses and the subjectivity of interpretation by operators. Moreover, if the test is combined with the Enzyme-Linked Immunosorbent Assay (ELISA) technique, the process is laborious. Here, we present a microneutralization assay that uses the PrestoBlue? reagent as a colorimetric CPE-based method. We evaluated the performance of this test on multiple strains of influenza virus by using human serum samples from a commercial panel and comparing both subjective (inverted microscope) and objective (spectrophotometer) readouts. Our results suggest that this colorimetric method for assessing CPE in microneutralization tests is more sensitive than the subjective method, while yielding comparable titers. This assay allows accurate and repeatable reading of the results, thereby reducing the possible uncertainty of the readout and speeding up the process.