Bioactive lipids are involved in cellular signalling events with links to human disease. Many of these are involved in inflammation under normal and pathological conditions. Despite being attractive molecules from a pharmacological point of view, the detection and quantification of lipids has been a major challenge. Here, we have optimised a liquid chromatography–dynamic multiple reaction monitoring–targeted mass spectrometry (LC-dMRM-MS) approach to profile eicosanoids and fatty acids in biological samples. In particular, by applying this analytic workflow to study a cellular model of chronic myeloid leukaemia (CML), we found that the levels of intra- and extracellular 2-Arachidonoylglycerol (2-AG), intracellular Arachidonic Acid (AA), extracellular Prostaglandin F2α (PGF2α), extracellular 5-Hydroxyeicosatetraenoic acid (5-HETE), extracellular Palmitic acid (PA, C16:0) and extracellular Stearic acid (SA, C18:0), were altered in response to immunomodulation by type I interferon (IFN-I), a currently approved treatment for CML. Our observations indicate changes in eicosanoid and fatty acid metabolism, with potential relevance in the context of cancer inflammation and CML.