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Estimating protein fractions and their degradation rate are vital to ensure optimum protein supply and degradation in the digestive system of ruminants. This study investigated the possibility of using the ANKOM gas production system and preserved rumen fluid to estimate the protein fractions and in vitro degradability of protein-rich feeds. Three in vitro methods: (1) gas production method (2) Cornell Net Carbohydrate and Protein System (CNCPS), and (3) the unavailable nitrogen assay of Ross (uN Ross ) were used to quantify protein fractions of four feeds (lupin meal, vetch grain, Desmanthus hay, and soybean meal). Rumen fluid mixed with 5% dimethyl sulfoxide and frozen at -20 °C was also compared against fresh rumen fluid in the gas production and uN Ross methods. All three methods ranked the feeds identically in the proportions of available (degradable or 'a + b') protein fractions as vetch grain, soybean meal, lupin meal, and Desmanthus hay in decreasing order. The use of fresh rumen fluid produced greater available protein fractions than preserved rumen fluid in all feeds. However, there was no difference between total gas production from lupin meal and vetch grain fermented for 16 h in either rumen fluid source. The in vitro degradable CP (IVDP) was higher for vetch grain (46 and 70%) at the 4th and 8th hours of incubation than other feeds, whereas soybean meal (85%) exceeded the other feeds after the 16th hour of incubation (P < 0.001). The greatest ammonia-N concentration was from soybean meal (1.27 mg/g) and lupin meal (0.87 mg/g) fermented for four hours using fresh rumen fluid. The proportion of fraction 'b' for soybean (82.1% CP) and lupin meals (39.4% CP) from the CNCPS method were not different (P = 0.001) from the fraction 'b' estimation of the gas production method for the same feeds (r = 0.99). Regardless of the methods, a greater water-soluble protein fraction was found from vetch grain (39.6-46.6% CP), and the proportion of fraction 'c' or unavailable protein in Desmanthus hay (39.1-41.5% CP) exceeded other substrates (P < 0.001). The strong positive correlation between fractions across different methods and identical ranking of feeds suggests the possibility of using ANKOM gas production apparatus for protein fractionation., (Copyright © 2023. Published by Elsevier B.V.)