Objective A simple and sensitive method for the determination of 3-nitropropionic acid (3-NPA) in plasma and urine by ion chromatography-triple quadrupole mass spectrometry (IC-MS/MS) was established. Methods Plasma and urine samples were extracted with aqueous solution containing 3% perchloric acid, and then the extract was centrifuged to remove the protein and lipids. The target analyte in the extract was cleaned-up by solid supported liquid/liquid extraction under pH=1-2,and methyl tert-butyl ether (MTBE) was used as eluent. After the MTBE was removed by nitrogen,the 3-NPA in the residues was dissolved into water. The separation of 3-NPA was carried out on a Dionex Ionpac AS19 analytic column (250 mm×2 mm, 7.5 μm) with gradient elution using KOH solution electrolytically generated from on-line eluent generation cartridge. Before the eluent flow entered the ion source of mass spectrometer,the potassium ion in the eluent was removed by an in-line suppressor. The 3-NPA was detected by negative electrospray ionization triple quadrupole mass spectrometry in the multiple reaction monitoring (MRM) mode. Results The result showed that the correlation coefficients of linear calibration cuve of 3-NPA were above 0.999 at the corresponding concentration ranges of 0.1-250 μg/L. The average recoveries were 92.0%-114% for 3-NPA in plasma and urine with relative standard deviations of 2.0%-12% (n=6). The limits of detection of 3-NPA(S/N=3)were 0.1 μg/L.Conclusion The method is simple, sensitive and accurate, and can be used for the determination of 3-NPA in plasma and urine.