Chronic low-grade inflammation is now considered to be central to the pathogenesis of various medical disorders like cardiovascular disease, atherosclerosis, hypertension, type 2 diabetes mellitus (T2DM) and cancer. The currently accepted model of inflammation proposes that both the induction of inflammation and its eventual resolution and return to homeostasis are coordinated, orderly processes actively signalled by specific protein and lipid molecules. Tissue damage activates the pro-inflammatory lipid mediators like prostaglandins and leukotrienes which are arachidonate (AA)-derived. Different lipid mediators such as lipoxin, resolvins and protectins, are generated during the resolution phase of inflammation, indicating that lipid mediator class switching takes place from the pro-inflammatory at the onset of inflammation to the pro-resolving at resolution. Whether deficiencies in the biosynthesis of lipid mediators might underlie some aspects of pathogenesis and complications in chronic inflammatory disorders is however completely unknown. We have developed a protocol for complete lipid mediator extraction from biological fluidics and complex tissue combined with a comprehensive liquid chromatography-tandem mass spectrometry methodology with a run time of 25 min/sample to identify and quantify 120 unique lipid species carried out within the framework of the Netherlands Metabolomics Centre. To demonstrate this strategy, we utilized this approach to investigate the first snapshot of lipid mediators in normal skin tissue.