Background Neisseria meningitidis is a cause of invasive bacterial disease associated with high morbidity. Immunization protects individuals against invasive meningococcal disease. The serum bactericidal assay (SBA) is required to evaluate functional activity of antibody produced in response to meningococcal vaccines. There are many assays to assess meningococcal SBA. We have improved in the SBA by using a dye, 2,3,5-triphenyl tetrazolium chloride (TTC) with a solid overlay agar method. Method Variant assay parameters such as target bacteria, baby rabbit complement, overlay agar and incubation time were optimized. Plate was overlaid with BHI agar containing TTC. Colorized colonies were counted using colony counting software. The standardization of SBA for meningococcal serogroup W-135 and Y were done with 50 pairs (pre- and post-immunized) of adult sera. Results Inter-assay reproducibility testing was achieved by coefficient of variation 15% (5% in serogroup W-135 and 2% in serogroup Y). The geometric mean titers were 44.5 (0.2 ~ 83.2, 95% CI) for serogroup W-135 and 28.7 (0.5 ~55.0, 95% CI) for serogroup Y in pre-immunized sera. After vaccination, subjects had significant increases in titers with 1,292.7 (0.5 ~ 2,466.9, 95% CI) of serogroup W-135 and 4,033.3 (0.6~6,434.0, 95% CI) of serogroup Y. The proportion of subjects with SBA titer ≥ 4-fold rises were 84% in serogroup W-135 and 96% in serogroup Y. Conclusion A solid agar overlay method reduces biohazards associated assay. Overlay agar with TTC permits high-throughput and high resolution imaging of bacterial colonies grown on plates without special equipment. This standardized SBA allows safe, easy, and efficient evaluation to measure immune response in meningococcal vaccines.