Plasma amino acid concentrations have been analyzed by high-performance liquid chromatography-electrospray ionization mass spectrometry (HPLC-ESI/MS) after precolumn derivatization. However, it is still unknown whether chyle and bilirubin, which are known to interfere with quantifications of some clinical biomarkers, have any influence on plasma amino acid measurement. In this study, we investigated the effects of those interference substances on amino acid concentrations determined by HPLC-ESI/MS. A total of 20 Japanese subjects (volunteers working for Ajinomoto Co., Inc.) were enrolled in this study. Twenty-one amino acid concentrations in plasma were measured with HPLCESI/MS after addition of each interference substances (a soy bean oil-egg yolk lecithin for the evaluation of chyle and total bilirubin). No significant changes of amino acid concentrations were observed by the addition of soybean oil (less than 0.25%) and bilirubin (final concentration: 0.5-2.0 mg/dL). Although the addition of 0.5% of soybean oil increased proline and phenylalanine levels significantly, the changes were less than ± 5% compared with blank samples. These results suggest that there would be little effects of chyle and bilirubin on the measurement of amino acid concentrations determined by the present HPLC-ESI/MS system.