Long chain fatty acids (LCFAs) function as a source of metabolic energy, substrates for membrane biogenesis, and storage of metabolic energy. Oxylipins, oxygenated derivatives of LCFAs, regulate the activity of many cellular processes. Existing methods for the analysis of LCFAs and oxylipins have limited compound coverage and sensitivity that, therefore, prevent their application in biological studies. In this work, we developed a high-throughput LC–MS method for analysis of 51 LCFAs and oxylipins. LCFAs and oxylipins were first extracted from biological samples via solid-phase extraction. The extracted molecules were analyzed by targeted comparative metabolomics. Saturated and monounsaturated LCFAs were analyzed in single ion reaction mode, while polyunsaturated LCFAs and oxylipins were analyzed in multiple reaction monitoring mode. Using this method, we successfully quantified 31 LCFAs and oxylipins from mouse livers.