Bile acids (BAs) are crucial for the diagnosis, follow-up, and prognostics of liver injuries and other BA metabolism related diseases. In particular, rodent unique BAs, α-muricholic acid (α-MCA), β-MCA, ω-MCA, tauro-α-MCA (α-TMCA), and β-TMCA, are valuable biomarkers for preclinical drug development. To the best of our knowledge, however, a simple, selective, sensitive, and robust analytical method for ω-MCA and taurine-conjugated MCAs has never been reported. We have developed a simple, selective, and sensitive analytical method for measurement of 16 BAs including the five rodent unique BAs in rat plasma using an ultra-performance liquid chromatography time-of-flight mass spectrometry (UPLC-TOF-MS) method. Activated charcoal was utilized to prepare BA-free plasma, which served as the surrogate matrix for the preparation of calibration standards and quality control (QC) samples. Results of matrix effects evaluation suggested that the BA-free plasma could be adequate as a surrogate matrix for BAs determination. Three stable isotope labelled internal standards were separated by reverse phase UPLC using gradient elution and were detected by TOF-MS in negative ion mode. The calibration curve was linear for all BAs over a range of 10-25ng/mL to 1000-10,000ng/mL, with overall imprecision below 15% and 20% at lower limit of quantification (LLOQ), respectively. This analytical method was used to determine BA concentrations in more than 300 plasma samples from rats with liver injuries induced using α-naphthylisocyanate, carbon tetrachloride, or flutamide. The alteration of BA concentrations was most evident for necrosis, and cholestasis hepatotoxins, with more subtle effects by steatosis and idiosyncratic hepatotoxins. In conclusion, we have developed a simple, selective, and sensitive analytical method to measure plasma 16 BAs including 5 rodent unique BAs, α-MCA, β-MCA, ω-MCA, α-TMCA, and β-TMCA. Our data suggested that α-TMCA and β-TMCA could be useful for identification or prediction of liver injuries, a currently unmet need in preclinical toxicity. Our method using TOF-MS is useful to determine BAs in rat plasma and of use in structural analyses of metabolites in early stage of drug development.