A new and robust high performance thin-layer chromatography (HPTLC) method has been developed for carbohydrate analysis in biorefinery streams. The samples can be applied directly from industrial streams without any purification or pretreatment, saving time and resources and avoiding potential alterations to the samples’ composition. The main neutral monosaccharides found in wood-based lignocellulosic biomass – glucose, xylose, mannose, arabinose and galactose – can be separated and quantified using acetonitrile:1-pentanol:water (4:1:1, v/v/v) on silica impregnated with a phosphate buffer. Common minor compounds – glucuronic acid, galacturonic acid, rhamnose, cellobiose and hydroxymethylfurfural (HMF) – can also be detected qualitatively. Matrix compounds, which are a major part of biomass, do not interfere with the analysis. Validation showed that the method is selective, has good repeatability and has limits of detection and quantification in the nanogram range. The visual fingerprint offers information on the samples’ constituents in the case of unknown samples. Also, incomplete hydrolysis is indicated by the visibility of oligosaccharides.