A procedure for the quantitative determination of gluten in wheat flour based on partial least squares (PLS) treatment of FT-Raman data is described. Results of similar quality were found using a PLS model derived from NIR (near infrared) spectra obtained in DRIFTS (diffuse reflectance infrared Fourier transform spectroscopy) mode and of slightly worse quality from the model constructed based on IR (infrared) spectra registered using a single reflection ATR (attenuated total reflection) diamond accessory. The relative standard errors of prediction (RSEP) were calculated for the calibration, validation and analysed data sets. These errors amounted to 3.2-3.6%, 3.5-3.8% and 4.8-5.7% for the three techniques applied, respectively. The proposed procedures can be used as simple, fast and accurate methods for the quantitative analysis of gluten in flour.