Using a semi-targeted approach, we have investigated the effect of acetaminophen on circulating bile acid profiles in rats, including many known bile acids and potential isomeric structures, as well as glucuronide and sulfate conjugates. The chromatographic separation was based on an optimized reverse-phase method exhibiting excellent resolution for a complex mix of bile acids using a solid-core C18 column, coupled to a high-resolution quadrupole time-of-flight system. The semi-targeted workflow consisted of first assigning all peaks detectable in samples from 46 known bile acids contained in a standard mix, as well as additional peaks for other bile acid isomers. The presence of glucuronide and sulfate conjugates was also examined based on their elemental formulae and detectable peaks with matching exact masses were added to the list of features for statistical analysis. In this study, rats were administered acetaminophen at four different doses, from 75 to 600 mg/kg, with the highest dose being a good model of drug-induced liver injury. Statistically significant changes were found by comparing bile acid profiles between dosing levels. Some tentatively assigned conjugates were further elucidated using in vitro metabolism incubations with rat liver fractions and standard bile acids. Overall, 13 identified bile acids, 23 tentatively assigned bile acid isomers, and 9 sulfate conjugates were found to increase significantly at the highest acetaminophen dose, and thus could be linked to drug-induced liver injury.