Fusarium head blight is a globally devastating fungal disease resulting in reduced grain yield and quality, along with contamination of grains with dangerous mycotoxins. Consumption of such mycotoxins by humans through processed food or livestock through feed has downstream implications of human and animal health. This interconnectivity across the environment, animal, and human health defines the One Health problem of threatened food safety and security. In this study, we explore remodeling of the wheat proteome upon exposure to a common mycotoxin, deoxynivalenol. We investigate cultivar specific responses to DON exposure in FHB-susceptible (Norwell) and -resistant (Sumai#3) cultivars across a continuum of exposure (i.e., 24 and 120 hours post inoculation), and upon low (i.e., 0.1 mg/mL) and high (1.0 mg/mL) levels of the mycotoxin. This complex experimental design enables us to tease apart the dynamic relationship between cultivar and DON tolerance. Specifically, we define specific proteins and broad categories of remodeling that are common (i.e., reduction in photosynthesis) and unique (i.e., glycosyltransferase) to the cultivars and align with anticipated protective mechanisms. Moreover, we applied an in vitro DON tolerance expression system and determined that induction of ubiquinol oxidase provides heightened protection for yeast growth relative to the negative control, as well as increased protection compared to a well-defined DON detoxifying protein. Our study suggests a new avenue for identification and characterization of novel DON detoxifying proteins as putative biomarkers for selected breeding strategies. Such strategies support the production of wheat varieties with increased tolerance to DON for improved global food safety and security.