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The study aimed to develop a bioassay for determining digestible reactive lysine in processed feedstuffs. The approach combines the determination of reactive lysine (guanidination method) in diets and digesta with a true ileal lysine digestibility assay. Using optimized reaction conditions, reactive lysine was determined in unheated casein and heated lactose/casein using both guanidination and FDNB methods. Similar values were obtained with both methods (0.53 and 0.32 mmol (g of casein)^-1 for casein and heated lactose/casein, respectively). Conventional lysine analysis overestimated (0.38 mmol g^-1) the reactive lysine in the heated mixture. The true ileal lysine digestibility in casein was 98.8%. The corresponding value in the heated lactose/casein was 70.5%, whereas the true ileal reactive lysine digestibility was 85.9%. The traditional approach to determining the digestible lysine in processed foods appears …