Nitrotyrosine (NTYR) is used as a biomarker of nitrative pathology caused by peroxynitrite (ONOO-) formation. NTYR measurement in biological materials usually employs such methodologies as immunohistochemistry staining, high-performance liquid chromatography and gas chromatography. In this study, we developed a method for the determination of tyrosine (TYR) and NTYR, using liquid chromatography with tandem mass spectrometry (LC-MS/MS). In order to confirm the applicability of our method to an in vivo system, we measured protein-bound NTYR levels using by LC-MS/MS method and immunohistochemical analysis in liver of B6C3F1 mice at 2 h, 4 h and 8 h after administration of 300 mg/kg acetaminophen (APAP). A mass spectrometer equipped with an electrospray ionization source using a crossflow counter electrode and ran in the positive ion mode (ESI+) was set up for multiple reaction monitoring (MRM), which monitored the transitions 182.2>136.2, 227.1>181.2, 191.3>144.4 and 236.3>189.5, for TYR, NTYR, [13C9]-TYR and [13C9]-NTYR, respectively. The average recoveries from mice liver protein samples spiked with 25 microM TYR and 100 nM NTYR were 94.4% and 95.6%, respectively, with correction using the added surrogate standards. The limits of quantification were 100 nM for TYR and 0.5 nM for NTYR. NTYR was detected all liver samples of mice by the proposed LC-MS/MS method. The concentration range of NTYR per milligram protein in samples was 0.17-0.3 pmol/mg protein. And the level reached a maximum at 4 h. These data were well correlated with the result obtained by an immunohistochemical reaction with anti-NTYR antibody. The LC-MS/MS method was able to determine protein-bound NTYR in a small amount of tissue sample, and is therefore expected to be a very powerful tool for evaluating ONOO- generation in an in vivo system.