The objective of this study was to estimate bioavailability of the third generation of a rumen-protected Lys (RPL) product, AjiPro-L (A3G; Ajinomoto Heartland Inc.) using the currently marketed AjiPro-L (A2G; Ajinomoto Heartland Inc.) after products were exposed to TMR. Fourteen multiparous lactating Holstein cows (114 ± 8 d in milk) housed in tie stalls were used in a replicated 7 × 7 Latin square design with 7-d periods. A common basal diet adequate in Lys was prepared 1 × /d and fed proportionately at 0500 h, 1300 h, and 2100 h. Treatments included Lys supplemented at 0, 75, 112.5 and 150 g/d from A2G and A3G and were mixed with a small amount of total mixed ration (TMR) once daily to mimic inclusion and exposure to a diet fed 1 × /d. Treatments were fed 3 × /d 1 h before feeding on d 2 through 7 of each period in amounts proportional to feed offered. Four blood samples were obtained from cows 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 AA concentrations. Data were reduced to period mean for each cow and analyzed using the MIXED procedure of SAS. The REG procedure was used to generate linear regression models for each RPL product using the values of Lys (µmol/L) and Lys as a percent of total AA (µmol/L basis) to determine the degree of elevation of plasma Lys in response to treatment. Relative to A2G, estimated bioavailability of A3G was determined using the slope-ratio assay technique. Plasma Lys was greatest (P ≤ 0.05) for 150 g/d A2G (119.8 ± 3.2 µmol/L) and 150 g/d A3G (123.8 ± 3.5 µmol/L) when compared with 0 g/d Lys (110.7 ± 3.3 µmol/L). The slope for A3G treatment was numerically greater (0.009; r² = 0.92) when compared with the slope for A2G treatment (0.006; r² = 0.93) when expressing the concentration of plasma Lys relative to total AA. Calculated bioavailability of A3G was 146.7% of the bioavailability of A2G. To our knowledge, this is the first study to measure relative bioavailability of RPL products in a TMR to simulate on-farm exposure when TMR is fed 1 × /d.