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High sensitive and precise analytical methods are needed in the modern bioprocess development and optimization of different biochemical and/or microbial technological steps of bioconversion of renewables. Investigation of polysaccharides in initial plant materials, as well as quantification of the products of bioconversion, improvement of the properties of new enzymes and fine tuning of biocatalytical processes can lead to ecologically friendly and cost-efficient alternatives to fossil-based technologies. Modern chromatography and mass-spectrometry methods are widely used in the analysis of simple sugars and plant polysaccharides, alcohols, organic and amino acids as microbial metabolites. These methods are extremely efficient in proteomic research, in biochemical properties and catalytic performance evaluation of new enzymes derived from natural and recombinant strains. Approaches for biological samples preparation followed by chromatographic separation and identification, and the methodology developed for the process control and optimization during carbohydrase strains cultivation will be discussed. The study of the main products of the enzymatic hydrolysis of plant polysaccharides, as well as minor amounts of side-products provides important information about the specificity and mechanisms of the enzymatic reactions. High sensitive methods with wide dynamic range based on HPAEX chromatography with pulse amperometric detection were optimized and applied in the analysis of mono- and oligosaccharides, derived for different sourses of biomass after chemical and/or enzymatic hydrolysis. Microbial synthesis of different chemical building blocks from biomass, e.g. oxy- and di-carbonic acids, based on fermentable sugars, is one of the important taskes for modern biotechnology. Rapid and similtaneous analysis of residual sugars, alcohols, and organic acids in the fermentation broth is crusial for the process development. Variation of selectivity of the high efficient ion-exchange resin by using different ratio of K+/Na+ followed by coupled diode-array and refractive index detection results in express and selective quanification of mono- and disaccharides (glucose, fructose, succrose), ethanol as well as fumaric and lactic asids.