Fusarium head blight (FHB), caused by Fusarium spp., is a destructive disease of small grain cereals, such as wheat, barley, oat and canaryseed. Apart from grain yield losses and reduced baking and seed quality, a major concern with FHB is crop contamination with Fusarium-produced trichothecene mycotoxins, specifically deoxynivalenol (DON), also known as vomitoxin. These mycotoxins accumulate in the grain making it unfit for consumption by humans and animals. Significant DON contamination may render a crop unmarketable, or reduces the market value by 40-65%. Breeding productive cultivars with high disease resistance and low mycotoxin contamination is a priority for wheat breeders. However, measurement of DON content is not always included in breeding programs due to the lack of efficient quantification methods. Some methods are easy-to-use, but they lack the needed accuracy and sensitive, such as enzyme-linked immunosorbent assay (ELISA); while some chromatographic-based methods have relatively higher accuracy and sensitivity compared to ELISA, but require complex extraction and cleanup steps and longer running time, which are not cost-efficient and environmentally friendly. In this study, we established a tandem mass spectrometry (MS/MS) method, which employed a one-step acetonitrile extraction protocol and flow injection analysis (FIA)-MS/MS method (i.e. no analytical column) to reduce the complexity, cost and time. This method is designed for FHB breeding programs or DON quantification for other purposes that require a fast, high-throughput DON phenotyping, but provides relatively high selectivity, accuracy and sensitivity compared to existing assays. This method has been fully validated according to the US Food and Drug Administration (FDA) Guidance for Bioanalytical Method Validation, including selectivity, linearity, accuracy, precision, recovery, matrix effects, stability and dilution integrity. With ease of use, high sensitivity and accuracy, this high throughput DON quantification method will increase breeding efficiency and accelerate the screening progress for FHB resistant germplasm.