Fusarium Head Blight (FHB) is a devastating disease commonly affecting North American small grains, primarily caused by Fusarium graminearum. Although the disease causes visible symptoms, the more pressing concern is the contamination of grain with trichothecene toxins, especially deoxynivalenol (DON), which can cause toxicity in humans and animals by inhibiting protein biosynthesis. The DON levels are more critical when breeding for resistance, but it does not account for pathogen infection and disease severity. To link DON and Fusarium infection, we optimized and validated a real-time multiplex quantitative PCR (qPCR) protocol for high-throughput quantification of Fusarium biomass based on the Tri5 gene, which encodes trichodiene synthase, the first enzyme involved in trichothecene toxin production. The primers and probes were designed to measure the abundance of Tri5 and normalized to the barley actin gene. The assay is repeatable, robust, and sensitive, up to 0.003 ng/ul of F. graminearum and 0.3 ng/ul of barley DNA. The method is cost-effective and has the potential to quantify Fusarium biomass, monitor disease progression, and determine DON levels in barley spikes and harvested grains.