In FHB infected cereals, Fusarium Damaged Kernels (FDKs) are the primary source of mycotoxin contamination, and even a small proportion of FDKs can elevate mycotoxin concentrations above regulatory limits. The percentage of FDKs is therefore widely used by plant pathologists and breeders as an indicator of FHB disease severity. In this project, we aim to develop a rapid and accurate LC-MS method to quantify major Fusarium-produced mycotoxins, including deoxynivalenol (DON), DON-3-glucoside (D3G), 3-Acetyl DON (3-AcDON) and 15-Acetyl DON (15-AcDON), at the individual kernel level. To establish the method, matrix-matched calibration curves were constructed for each toxin using barley and wheat kernel matrices; limits of detection (LOD), limits of quantification (LOQ), and analytical recoveries were rigorously validated. Finally, the method was applied to single kernels from a barley spike and a wheat spike to identify and spatially locate FDKs along the spike. This pipeline enables pathologists and breeders to accurately assess FHB incidence and spike-level disease progression in research applications.