Rumen-protected (RP) Met, Lys, and His have been added to diets for optimizing milk protein synthesis and reduce N excretion to the environment. Therefore, it is critical to accurately estimate the relative bioavailability (RBV) of RPAA supplements. The plasma free AA dose-response technique has been used to determine RB of RP-Met and RP-Lys, but not RP-His. Our objective was to determine the RBV of a prototype RP-His supplement (Ajinomoto Co., Inc., Japan) using the plasma AA dose-response method. Six multiparous Holstein cows fitted with ruminal cannulas were used in a 6 × 6 Latin square design with 7 d experimental periods. The basal diet consisted (DM basis) of 31% corn silage, 20.4% haylage, and 48.6% concentrate. The diet was 16.4% CP, 177 g/d MP, and 3.5 Mcal/d NE_L. Treatments were abomasal infusions of His at 0, 8, 16 and 24 g/d, and 20.2 and 33.6 g/d of RP-His (42% His in the prototype). Blood samples were collected at 2, 4, 6, and 8 h after the morning feeding in the last 3 d and composited by day. Plasma AA was quantified using ultra performance liquid chromatography tandem mass spectrometry. Data were analyzed with the MIXED and PROC REG procedures of SAS. Both DMI and milk yield were not affected by treatments. Plasma His concentration linearly increased from 49.1 to 79.0 µM (P < 0.0001) in response to incremental amounts of His infused in the abomasum. Feeding RP-His also elevated the concentration of plasma His linearly (49.1 to 66.8 µM, P < 0.0001). Plasma carnosine concentration showed linear responses to both abomasal infusion of His (14.4 to 16.3 µM, P = 0.017) and RP-His supplementation (14.4 to 16.2 µM, P = 0.043). Treatments did not significantly change the concentrations of other plasma AA. The slopes from regressing plasma concentration of His (%TAA minus His) on incremental amounts of abomasally infused His or RP-His averaged 0.0463 and 0.0225, respectively. The RBV of the prototype RP-His tested averaged 48.6 ± 3.24 (0.0225 divided by 0.0463 times 100). Our data suggest that approximately 48% of His in RP-His was absorbed in the small intestine when using the plasma free AA dose-response technique.