The plasma free AA dose-response method has been used to determine the relative bioavailability of AA in encapsulated rumen-protected AA supplements The objectives of this experiment were to determine which of the sulfur-containing plasma metabolites would respond in a positive linear fashion to increasing amounts of absorbed dl-2-hydroxy-4-methylthiobutanoic acid (HMTBa) and which expressions of metabolite concentrations would provide the greatest precision of measurement. Five mid-lactatation multiparous Holstein cows, equipped with ruminal cannulas and fed a Met-deficient diet, were assigned to a 5 × 5 Latin square with 7-d experimental periods. Treatments were daily abomasal infusions of 0, 6, 12, 18 and 24 g/d of HMTBa. Blood samples were taken from the tail vein the last 3 d of each period at 2, 4, 6 and 8 h after the morning feeding, cooled, and then centrifuged. One sample of plasma was deproteinized for AA analysis and one sample of plasma was retained for HMTBa analysis. Daily composite samples from the 4 sampling times for each cow were stored at −80°C for later analysis. Production and plasma data were analyzed using the PROC MIXED and PROC REG procedures of SAS 9.4. Statistical significance was declared at P ≤ 0.05. Intake of DM and milk yield were not affected by treatments. Increasing amounts of HMTBa resulted in linear increases in milk protein concentrations (P = 0.03) and plasma concentrations of HMTBa, Met, cystathionine/allocystathionine, total S-AA plus HMTBa (P < 0.001) and total S-AA (P = 0.009). As indicated by lower CV and higher r² values, precision of the described linear relationships for plasma metabolites were improved by expressing their concentrations as percentages of total AA. There were no effects of treatments on other plasma AA. We conclude the plasma free AA dose-response method can be used in future experiments to determine the relative Met bioavailability of Met analogs fed to lactating dairy cows.