The clinical importance of prostaglandins and leukotrienes in asthma is well recognized; however, the biochemical role of other lipid mediators (often termed oxylipins) in the regulation of airway tone and inflammation remains unclear. We therefore developed a workflow to investigate oxylipin physiology and pharmacology in two in vitro models, the intact human bronchus and the guinea pig trachea. Airways were isolated and smooth muscle contraction was measured in an organ bath following stimulation with either anti-IgE or ovalbumin. The associated release of oxylipins over time into the organ bath was quantified using three developed LC-MS/MS methods capable of collectively measuring 130 compounds. Oxylipin extraction recoveries were 71% on average, method accuracy was 90-98%, coefficient of variation was 4.3-9.4%, and matrix effects were on average 11%. At baseline, low levels of primarily prostaglandins and associated metabolites were observed in both tissue preparations. The mast cell-induced airway constriction caused release of leukotrienes and further elevations in prostaglandin levels. In total, 57 oxylipins from the human bronchus, and 42 from guinea pig trachea, were detected at 60 min post-stimulation in the organ bath. Chiral analysis demonstrated that 5-hydroxyeicosatetraenoic acid (5-HETE) in the human bronchus preparation was not produced by 5-LOX enzymatic activity (enantiomeric excess [ee] = 10%), as opposed to 12( S)-HETE, 14( S)-, and 17( S)-hydroxy docosahexaenoic acid (HDoHE; ee = 100%), highlighting that chiral chromatography is necessary for correct biological interpretation. Unexpectedly, prostaglandin D2 and its metabolites remained elevated 24 h after the challenges, suggesting a sustained activation of mast cells not previously described. The reported translational methodology provides a new platform for comprehensive studies to elucidate the origin and functions of individual oxylipins in various airway responses.