Introduction: A resurgence of mumps among fully vaccinated adolescents and young adults globally has led to questions about the longevity of vaccine derived specific immunity. Unfortunately, the ideal serological correlate of immunity to mumps has yet to be identified. However, neutralising antibody titres in serum are used extensively as a surrogate marker of immunity to mumps. Conventional neutralisation tests are technically challenging, thus we developed and validated a high throughput, RT-qPCR microneutralisation (RT-qPCR-MN) method to determine serum neutralising antibody levels to mumps virus strains which avoids a number of the technical limitations of existing methods. Methods: The qPCR-MN assays were thoroughly validated using human serum samples from patients with prior exposure to mumps infection or vaccination. Results: Each sample of pooled sera neutralised virus at a constant rate and without significant changes when tested against genotype A (MuV-A) and G (MuV-G) mumps virus concentrations from 200 to 3200 TCID50. The within run and between run variation of the RT-qPCR-MN assays for both genotypes were less than 3 % and 9 % for low and high titre samples, respectively. The correlation between the focus reduction neutralisation test and RT-qPCR-MN was excellent for both MuV-G (r2 = 0.80, 95CI: 0.67–1.00, p < 0.0001) and MuV-A genotypes (r2 = 0.88, 95 %CI 0.69–1.00, p < 0.0001) endpoint determinations. Conclusions: We have developed a RT-qPCR MN assay for mumps virus that is simple, fast, scientifically objective and has high throughput. The assay can be used to provide key insights into the efficacy of mumps vaccination, to help explain the causes for the resurgence of mumps infection in vaccinated populations.