This study explores the impact of two inoculation methods, dip and spray, on FHB severity in transgenic barley lines under controlled conditions. The dip method occasionally resulted in unfilled or non-infected grains, prompting an investigation whether DON is consistently associated only with visibly infected grains or whether additional variables are at play in influencing DON levels. Results revealed a highly significant difference in FHB severity between the dip and spray methods, emphasizing the critical role of the inoculation technique. The 95% confidence interval indicated severity ranges of 61.5% to 71.5% for the spray method and 52.11% to 57.40% for the dip method. Notably, the dip method presented a 7% chance of reaching 100% severity would give a potential method for specific study purpose. Fusarium-Damaged Kernels (FDK) percentages varied considerably among spikes within genotypes for both methods, with the dip method exhibiting greater sample severity variation. Neither inoculation method significantly affected deoxynivalenol (DON) levels. However, both methods revealed significant variation in DON levels across multiple spikes within each line. Further analysis focused on DON levels and kernel properties from the dip inoculation study. Regression analysis indicated significant influences of kernel types on DON levels, with the proportion of uninfected and infected kernels serving as highly significant predictors. To ascertain the contribution of asymptomatic and/or unfilled kernels to DON levels, experiments on individual kernels are recommended. A thorough understanding of DON contamination dynamics can be obtained by analyzing DON for each uninfected or unfilled grain separately. This research aims to contribute valuable insights into the complex dynamics influencing DON levels in barley, paving the way for more effective FHB resistance strategies.