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Objective The objective of this study was to determine internal structure spectral profile of by-products from coffee processing that were affected by added-microorganism fermentation duration in relation to truly absorbed feed nutrient supply in ruminant system. Methods The by-products from coffee processing were fermented using commercial fermentation product, consisting of various microorganisms: for 0 (control), 7, 14, 21, and 28 days. In this study, carbohydrate-related spectral profiles of coffee by-products were correlated with their chemical and nutritional properties (chemical composition, total digestible nutrient, bioenergy values, carbohydrate sub-fractions and predicted degradation and digestion parameters as well as milk value of feed). The vibrational spectra of coffee by-products samples after fermentation for 0 (control), 7, 14, 21, and 28 days were determined using a JASCO FT/IR-4200 spectroscopy coupled with accessory of attenuated total reflectance (ATR). The molecular spectral analyses with univariate approach were conducted with the OMNIC 7.3 software. Results Molecular spectral analysis parameters in fermented and non-fermented by-products from coffee processing included structural carbohydrate, cellulosic compounds, non-structural carbohydrates, lignin compound, CH-bending, structural carbohydrate peak1, structural carbohydrate peak2, structural carbohydrate peak3, hemicellulosic compound, non-structural carbohydrate peak1, non-structural carbohydrate peak2, non-structural carbohydrate peak3. The study results show that added-microorganism fermentation induced chemical and nutritional changes of coffee by-products including carbohydrate chemical composition profiles, bioenergy value, feed milk value, carbohydrate subfractions, estimated degradable and undegradable fractions in the rumen, and intestinal digested nutrient supply in ruminant system. Conclusion In conclusion, carbohydrate nutrition value changes by added-microorganism fermentation duration were in an agreement with the change of their spectral profile in the coffee by-products. The studies show that the vibrational ATR-FT/IR spectroscopic technique could be applied as a rapid analytical tool to evaluate fermented by-products and connect with truly digestible carbohydrate supply in ruminant system.