Targeted proteomic mass spectrometry is emerging as a salient clinical diagnostic tool to track protein biomarkers. Yet, its strong analytical properties have not been exploited in the diagnosis and typing of flavivirus. Here, we report the development of a highly sensitive and specific single-shot robust assay for flavivirus typing and diagnosis using targeted mass spectrometry technology. Our flavivirus parallel reaction monitoring assay (fvPRM) has the ability to track secreted flaviviral non-structural protein 1 (NS1) over a broad diagnostic and typing window with high sensitivity, specificity, extendibility and multiplexing capability. These features, pivotal and pertinent to efficient response towards flavivirus outbreaks, including newly emerging flavivirus strains, and circumvent the limitations of current diagnostic assays. fvPRM thus carries high potential in positioning itself as a forerunner in delivering early and accurate diagnosis for disease management.