Attenuated total reflectance Fourier transform mid-infrared (ATR-FTIR) spectroscopy is widely applicable for the chemical analysis of biological materials, relatively inexpensive, requires only simple sample preparation, and is of comparatively high-throughput compared to traditional wet chemical or chromatographic methods. It is particularly well suited for the non-destructive analysis of dried and finely ground plant samples for the subsequent prediction of cell wall and other compositional or processing parameters using chemometric regression models. Furthermore, analysis of mid IR spectra by non-regression methods e.g. principal components analysis provides a straight forward approach for multivariate comparison of the effects of experimental, processing, and environmental treatments, and genotypic and temporal differences on chemical composition including changes in cell wall composition. There is thus great potential for using ATR-FTIR in the lignocellulosic biomass industry at a number of levels. Here we describe methods for cell wall sample preparation, generation of ATR-FTIR spectra, and suggest techniques for the statistical analysis and/or chemometric pattern recognition between the analysed samples.