Summary: Estimating the relative bioavailability of rumen-protected (RP) amino acid supplements using the plasma free amino acid dose-response methodology currently uses blood collected at 2, 4, 6, and 8 hours after the 0500 hour feeding over 3 days, with cows being fed every 8 hours (0500, 1300, and 2100 hours). We conducted a study where blood samples were collected at 2, 4, 6, and 8 hours after each of the feedings to investigate whether this sampling protocol captures the diurnal changes in plasma methionine (Met) concentrations that may exist to adequately calculate the relative bioavailability of Met from RP-Met supplements. There was no diurnal variation in plasma Met concentration, and the relative bioavailability calculated for the RP-Met supplement tested did not differ significantly, thus indicating that the proposed sampling protocol can be used to determine the relative bioavailability of RP-Met supplements.