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To our knowledge, there is limited study on the relationship between the molecular structure of feed and nutrient availability in the ruminant system. The objective of this study is to use advanced vibrational molecular spectroscopy (attenuated total reflection [ATR]-Fourier transform infrared [FT/IR]) to reveal carbohydrate molecular structure properties of faba bean partitions (stem, leaf, whole pods [WP], and whole plant) and faba bean silage before and after rumen incubation in relation to nutrient availability and supply to dairy cattle. The study included the correlation between carbohydrate-related spectral profiles and chemical profiles, feed energy values, Cornell Net Carbohydrate and Protein System carbohydrate fractions, and rumen degradation parameters of faba bean samples (whole crop, stem, leaf, WP, and silage) before and after rumen incubation. FTIR spectra of faba bean sample before and after 12 and 24 h rumen incubations were collected with JASCO FT/IR-4200 with ATR at mid-IR range (ca. 4000-700 cm -1 ) with 128 scans and at 4 cm -1 resolution. The univariate molecular spectral analysis was carried out using OMNIC software. The results show that ATR-FT/IR spectroscopic technique could detect the change of microbial digestion to carbohydrate-related molecular structure. The spectral parameters of feed rumen incubation residues had a stronger correlation with less degradable carbohydrate fractions (neutral detergent fiber, acid detergent fiber, acid detergent lignin, hemicellulose, and cellulose) while spectral profiles of original faba samples had a stronger correlation with easily degradable carbohydrate fractions (starch). In conclusion, rumen degradation of carbohydrate contents can be reflected in the change of its molecular spectral profiles. The study shows that vibrational molecular spectroscopy (ATR-FT/IR) shows high potential as a fast analytical tool to evaluate and predict nutrient supply in the ruminant system., (© 2022 Wiley-VCH GmbH.)