Targeted proteomic mass spectrometry is emerging as a salient clinical diagnostic tool to track protein biomarkers. However, its strong analytical properties have not been exploited in the diagnosis and typing of flaviviruses. Here, we report the development of a sensitive and specific single-shot robust assay for flavivirus typing and diagnosis using targeted mass spectrometry technology. Our flavivirus parallel reaction monitoring assay ( fv PRM) has the ability to track secreted flaviviral nonstructural 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 toward flavivirus outbreaks, including newly emerging flavivirus strains, circumvent the limitations of current diagnostic assays. fv PRM thus carries high potential in positioning itself as a forerunner in delivering early and accurate diagnosis for disease management.