A study of NIR as a tool for process monitoring of thermophilic anaerobic digestion boosted by glycerol has been carried out, aiming at developing simple and robust Process Analytical Technology modalities for on-line surveillance in full scale biogas plants. Three 5 L laboratory fermenters equipped with on-line NIR sensor and special sampling stations were used as a basis for chemometric multivariate calibration. NIR characterisation using Transflexive Embedded Near Infra-Red Sensor (TENIRS) equipment integrated into an external recurrent loop on the fermentation reactors, allows for representative sampling, of the highly heterogeneous fermentation bio slurries. Glycerol is an important by-product from the increasing European bio-diesel production. Glycerol addition can boost biogas yields, if not exceeding a limiting 5-7 g L-1 concentration inside the fermenter-further increase can cause strong imbalance in the anaerobic digestion process. A secondary objective was to evaluate the effect of addition of glycerol, in a spiking experiment which introduced increasing organic overloading as monitored by volatile fatty acids (VFA) levels. High correlation between on-line NIR determinations of glycerol and VFA contents has been documented. Chemometric regression models (PLS) between glycerol and NIR spectra needed no outlier removals and only one PLS-component was required. Test set validation resulted in excellent measures of prediction performance, precision: r(2) = 0.96 and accuracy = 1.04, slope of predicted versus reference fitting. Similar prediction statistics for acetic acid, iso-butanoic acid and total VFA proves that process NIR spectroscopy is able to quantify all pertinent levels of both volatile fatty acids and glycerol.