The wet milling process can be a valuable alternative for deoxynivalenol (DON) contaminated wheat grain, taking advantage of DON water solubility. The present research was conducted on hard red spring wheat (HRSW) and durum wheat (DW) samples contaminated with DON. The wheat samples were dry milled into farina and semolina, which were further subjected to three different small-scale wet milling tests, dough-washing (Martin process), moderately sheared dough-water dispersion (medium shear) process, and highly sheared flour-water dispersion (high shear) process. Gluten samples extracted from all the wet milling processes had DON levels below the limit of quantification (<0.2 mg/kg) because of the high water solubility of DON. Size-exclusion HPLC revealed that gluten samples obtained from medium shear and high shear processes were composed of more unextractable (sonication extractable) glutenin macropolymers and less gliadin fractions than those obtained from the Martin process. This yielded better rheological performance for the gluten samples, so that they were quite similar to the control (commercial wheat gluten), which means the gluten samples exhibited a potential application as food ingredients.