Complexity and high cost are the main limitations for high-throughput screening methods for the estimation of the sugar release from plant materials during bioethanol production. In addition, it is important that we improve our understanding of the mechanisms by which different chemical components are affecting the degradability of plant material. In this study, Fourier transform infrared photoacoustic spectroscopy (FTIR-PAS) was combined with advanced chemometrics to develop calibration models predicting the amount of sugars released after pretreatment and enzymatic hydrolysis of wheat straw during bioethanol production, and the spectra were analysed to identify components associated with recalcitrance.