Polymeric material derived from gluten has become competitive as an alternative to synthetic polymers due to its low cost, good mechanical properties and renewable source. The study of rheological characteristics of gluten-glycerol mixtures is of great importance for the suitable use of these materials. The aim of the present study was to investigate the viscoelastic behavior of gluten plasticized with glycerol at different process conditions. Mechanical spectra of gluten and glycerol mixtures in different proportions (1:0.6, 1:1.0 and 1:1.4, respectively) were determined by scanning frequency in the range of 0.1 - 30 Hz, at constant stress (30 Pa) and temperature (25 °C) using a rheometer AR1500ex (TA Instruments, Elstree, UK). The effect of thermal treatment at 40 °C and 70 °C was also evaluated. All the mixtures produced with different glycerol content and temperatures presented a gel-like behavior, with G’>G’’. Regardless of the concentration of glycerol, the increase in the process temperature increases the storage and loss modulus of the mixtures. These changes can be explained by the fact that the thermal treatment affects the structuring of the material, favoring the formation of covalent cross-links, probably via disulfides bonds. For the other hand, an inverse rheological behavior of these mixtures was observed by the increase of glycerol content, since the storage and dissipation moduli decreased with higher concentrations of this plasticizer. Glycerol content affected the hydrogen bonds, increasing the water content in the material. This increase in the interactions between the polymer and the water reduces the density of physical junctions, consequently reducing the storage modulus. Accordingly, it was observed that the rheological behavior of the plasticized gluten was sensitive to temperature variation and concentration of plasticizers. Thus, these variables must be used according to the characteristics expected for the application of this material.