High-quality analysis of pretreatment outcome, cost-effective enzymatic hydrolysis and discovery of new efficient enzymes are needed in order to obtain commercially viable production methods for 2G bioethanol. GlycoSpot has already demonstrated that multi-colored chromogenic polysaccharide and protein substrates can be used in cheap, convenient and high-throughput multiplexed assays. In addition, we have produced substrates out of biomass materials in which the complexity of plant cell walls is partially maintained. These substrates can be used to screen the activities of different carbohydrate-acting enzymes and provide insight into substrate availability within biomass. However, these substrates do not allow screening for enzymes where the fine structure of the substrate is required -for example esterases and exo-acting enzymes. We aim to develop the next generation of biomass substrates in which native structure are minimally modified and chemical substitutions are maintained. These novel True-To-Nature Complex Biomaterial Substrates (TTN-CBS) would more closely mimic natural biomass encountered in biorefining and new findings can be directly applied to improvement of raw material utilization.