Quantification of carbohydrates and metabolites in fermentation processes can be considered as key factor in determining yield and productivity for a better understanding of the microbial behavior under different conditions. The main aim of the present study was to develop and set up analytical methods for detecting complex sugar and/or metabolite mixtures in fermentation broth based on high-performance thin-layer chromatography (HPTLC). HPTLC is a fast and accurate method of separating complex mixtures, based on planar development. The proposed methods involved the separations of a mixture of monosaccharides (glucose, xylose, arabinose, and rhamnose) deriving from delignification and hydrolysis of hazelnut shells and the corresponding sugar alcohols (xylitol, arabitol, and sorbitol) obtained by fermentations of Candida tropicalis spp., a yeast able to ferment aldoses to produce sugar alcohols. HPTLC methods were set up on simple chamber development and using instrumental techniques like overpressured layer chromatography (OPLC). This approach has enabled the simultaneous monitoring of several samples with significant time and money savings. Different multicomponent broths at different times of fermentation were analyzed.