Lysine bioavailability of second generation AjiPro-L (A2G; Ajinomoto Heartland, Inc.) has been determined using the plasma free amino acid dose response technique. The objective of this study was to compare bioavailability of MetaboLys (MBL; H. J. Baker & Bro., Inc.) and USA Lysine (USA; Kemin Industries Inc.) relative to A2G following the same technique. Fourteen multiparous lactating Holstein cows (113 ± 28 d in milk) were housed in a tie stall facility and used in a replicated 7 × 7 Latin square design with 7-d periods. Cows were blocked by days in milk and milk production, and assigned randomly to treatment sequence. A common basal diet adequate in Lys was fed proportionately at 3 intervals (33.4% at 0500 h, 33.3% at 1300 h, and 33.3% at 2100 h). Treatments included Lys supplemented at 0, 30, or 60 g/d from A2G, MBL, or USA and were administered 1 h before each feeding on d 2 through 7 of each period in amounts proportional to feed offered to simulate diet inclusion. Blood samples were obtained from each cow on d 6 and 7 of each period from the tail vein at 2-h intervals starting at 0600 h. Plasma, pooled by day, was analyzed for amino acid (AA) concentrations. Data were reduced to a period mean for each cow and analyzed using the MIXED procedure of SAS. REG procedure was used to generate linear regression models for each rumen-protected Lys product using the values of Lys (µmol) and Lys as a percentage of total AA (µmol-basis) to determine the degree of elevation of plasma Lys. Relative to A2G, estimated bioavailability of MBL and USA were determined using the slope ratio assay technique. Dry matter intake and milk yield (P > 0.10) did not differ among treatments. The slope for A2G was numerically greater (0.183; r² = 0.72) when compared with the slope for MBL (0.03; r² = 0.58) or USA (0.07; r² = 0.99) when expressing the concentration of plasma Lys as absolute value (µmol). Calculated bioavailability of MBL and USA was 18.3 and 38.2% of the bioavailability of A2G, respectively. Characterizing relative bioavailability of rumen-protected Lys in dairy cattle provides a means for comparing economic value of commercially available products.