In nutrition studies, para-aminohippuric acid (PAH) is a marker frequently used to measure blood flow in pigs, which is essential for estimating portal-drained viscera (PDV) flux of nutrients. The aim of this study was to evaluate the PAH analytical method by means of qualimetric statistical procedures to estimate the matrix effect and the accuracy and limits of quantitation of the method. Net PDV flux of nutrients was determined in five multi-catheterized pigs using water, plasma or commercial serum as standard matrix. A proportional systematic error due to matrix effect was found for plasma and serum. Mean recovery was 99.4%, and intra- and inter-day precision of the method was 2.4% and 3.8% relative standard deviation, respectively. The limit of quantification was 0.22 mg PAH/l. Use of water for the PAH standard curves underestimated portal blood flow compared with PAH standards prepared with plasma or commercial serum (706, 954 and 927 ml/min; P<0.05, respectively). Consequently, PDV O2 consumption, glucose and amino acids fluxes were underestimated by 33% (P<0.001). In conclusion, our results stress the importance of using plasma from pigs not infused with PAH or alternatively commercial pig serum to prepare PAH standards to determine blood flow in pigs to avoid underestimation of blood flow.