Interaction of hydrocolloids (xanthan gum, locust bean gum, guar gum, and high-methoxyl pectin) with macrocomponents of dough (water, starch, and protein) was evaluated by different techniques. (1)H spin-spin NMR relaxation assays were applied to study the mobility of the gluten-hydrocolloid-water matrix, and the amount of freezable water was determined by differential scanning calorimetry (DSC). Starch gelatinization parameters (T, enthalpy) were also analyzed by DSC. The influence of additives on the protein matrix was studied by Fourier transform (FT) Raman assays; analysis of the extracted gliadins and glutenins was performed by electrophoresis (SDS-PAGE). A significantly higher molecular mobility was found in matrices containing xanthan gum, whereas pectin led to the lowest molecular mobility. Freezable water showed a trend of increasing in the presence of hydrocolloids, particularly under conditions of water restriction. Starch gelatinization final temperature was decreased when hydrocolloids were added in the presence of enough water. In general, FT-Raman and SDS-PAGE indicated that hydrocolloid addition promoted a more disordered and labile network, particularly in the case of pectin addition. On the other hand, results obtained for dough with guar gum would indicate a good compatibility between this hydrocolloid and the gluten network.