Technical Abstract: An infrared spectroscopic method was developed to determine changes in the secondary structure of gluten proteins in a flour-water dough system as it was mixed. FT-HATR mid-infrared spectra of mixed doughs revealed changes in four bands in the amide III region typically associated with secondary structure of proteins: 1317 (a-helix), 1285 (b-turn), 1265 (random coil), and 1242 cm-1 (b-sheet). The largest band, which also showed the greatest change in second derivative band area (SDBA) during mixing (increasing over time), was the band at 1242 cm-1. The bands at 1317, 1285, and 1242 cm-1 also showed an increase in SDBA over time. Alternatively, the band at 1265 cm-1 showed a corresponding decrease during mixing. All bands reached an optima (or minima) corresponding to the optimum development of the dough. Increases in a-helical, b-turn, and b-sheet structures during mixing suggest that the secondary structure of gluten protein assumes a more ordered conformation, apparently at the expense of random coil structure in the macromolecule. These results demonstrate that it is possible to follow the rheological behavior of dough based on changes in the protein structure of the system.