Para-aminohippuric acid (pAH) is a marker frequently used to measure plasma or blood flow. In sheep studies, it is recognized that its determination must include a deacetylation step to counteract the hepatic acetylation of pAH. Such a procedure is not of common usage in cattle studies although a recent suggestion of hepatic pAH acetylation in dairy cows may have important consequences for hepatic nutrient fluxes. The aims of this study were to evaluate pAH analytical methods according to international procedures of metrology and confirm hepatic acetylation of pAH in dairy cows. The effect of the matrix used to prepare the standard curve was tested, and the influence of the pAH analytical method on blood flows and subsequent net hepatic fluxes of nutrients was determined. For the first objective, accuracy profiles were established and bias, indicators of precision, and limits of quantification were reported for 2 analytical methods (without and with a pAH deacetylation step) using 2 different standard matrices (water and plasma). Second, the net hepatic flux of different nutrients was determined including or not the deacetylation step and preparing the standard curves in plasma using samples collected from 5 multicatheterized lactating Jersey cows. The choice of the matrix had a significant impact on plasma pAH concentrations as illustrated by accuracy profiles. Water matrix decreased (P < 0.01) the slope, y-intercept, and the absorbance at concentration 0 mg pAH/L of the standard curve in both methods (without and with the deacetylation), supporting that standards prepared in water should not be used to analyze plasma samples. Samples collected on cows confirmed hepatic acetylation of pAH across the liver. Deacetylation, performed using plasma as the standard matrix, increased (P < 0.05) plasma pAH concentrations from 18.4, 26, and 23.5 to 21.4, 28.9, and 27.3 mg/L in the artery, portal vein, and hepatic vein, respectively. Deacetylation decreased the hepatic venous and arterial plasma and blood flows (P < 0.05) by 9 and 55%, respectively, modifying the net hepatic flux of acetate, total amino acid, and oxygen by more than 19% (P < 0.05). In conclusion, our results highlight the importance of including a deacetylation step in the pAH analysis method in cattle studies and of using plasma as standard matrix