Lysine (Lys) availability in three different soya-bean meal (SBM) products was determined using the following techniques: whole body (WB) net flux of Lys, digestible Lys (duodenal flow × intestinal digestibility) and the plasma Lys response curve method of Rulquin and Kowalczyk (2003). Four multiparous Holstein cows (173 days in milk) were equipped with ruminal and duodenal cannulas and used in a 4 × 4 Latin square experiment with 14-day periods. The animals were fed either solvent-extracted SBM (SE), expeller-processed SBM (EP) or lignosulphonate-treated SBM (LS) at 23% of the diet dry matter (DM). The fourth treatment (SE70) consisted of a continuous infusion of Lys (70 g/day) into the omasum of cows fed the SE diet. Chromium(III) oxide was included as a digesta marker in order to determine the duodenal flow of amino acids (AA). On day 12 of each experimental period, six blood samples were collected to determine plasma Lys concentrations. Immediately after that, a pulse dose of L-[2–15N] Lys was administered in the jugular vein. Jugular blood samples were then collected at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 13, 16, 19, 25 and 31 min after the injection to determine 15N Lys enrichment. On each of days 13 and 14, eight digesta samples were collected and pooled by period. Amongst the diets of SBM (SE, EP, LS), no differences were observed for duodenal Lys flow or digestible Lys. Duodenal flow of microbial N with SE was numerically higher, compared with EP and LS, indicating enhanced duodenal supply of microbial Lys for this diet, and this may have compensated for the additional Lys derived from undegradable protein in rumen-protected SBM products (EP and LS). The use of the plasma response curve method as well as the measurement of WB Lys flux also revealed no differences in Lys availability among the SBM products. The WB flux method resulted in 100% post-ruminal recovery of the Lys infused with diet SE70 compared with the control diet SE, which indicates that the method is reliable for determining Lys availability. The Lys flux approach not only allows for the estimation of intestinally available essential AA but also it avoids the use of cannulated animals.