Abstract The cytochrome P450 metabolites of arachidonic acid (AA) are mostly present in tissues, such as the liver, as bound to phospholipids, with only a small fraction available as free acids. The purpose of this study was to develop and validate a UHPLC–MS/MS method for quantitation of free liver concentrations of AA and four epoxygenated (5,6-, 8,9-, 11,12-, and 14,15-EET), four dihydroxylated (5,6-, 8,9-, 11,12-, and 14,15-DHET), and two ω/(ω−1) hydroxylated (19- and 20-HETEs) metabolites of AA in rat livers using deuterated internal standards. The analytes were rapidly and efficiently (79–92%) recovered from 100mg of fresh liver into methanol. After evaporation, the reconstituted samples were injected either undiluted (for the simultaneous analysis of the metabolites) into a gradient or diluted (for AA analysis) into an isocratic UHPLC system with run times of 5 and 2min, respectively. Mass spectrometry was conducted using multiple reaction monitoring in negative mode. The method was linear (r2≥0.98) in the concentration ranges tested for metabolites (0.19–120ng/g liver) and AA (7.8–500μg/g liver). The lower limit of quantitation of the assay was between 0.57 and 5.6pg injected on column for different AA metabolites. The assay was validated (n=5) based on acceptable intra- and inter-run accuracy and precision values. Additionally, matrix effect was minimal for most analytes. Freeze-thaw of samples drastically increased the free liver concentrations of analytes, presumably due to their release from the membrane storage sites. Therefore, fresh liver samples should be used for analysis. However, the methanolic extracts may be stored at −80°C for at least two weeks without any compromise. The method was successfully used in the measurement of all the analytes in the rats subjected to 60min of hepatic ischemia (n=6) or sham operation (n=6). Ischemia resulted in significantly higher free concentrations of AA and most of its studied metabolites. The method is precise, accurate, and sensitive for measurement of free liver concentrations of AA and its P450 metabolites in the rat liver.