Chronic inflammation is associated with many diseases including rheumatoid arthritis, asthma, diabetes, inflammatory bowel disease and neurological diseases such as Alzheimer’s disease (Serhan, 2014). Lipid mediators, derived from the oxygenation of polyunsaturated fatty acids (PUFAs) through enzymatic or free-radical mediated mechanisms, are implicated in the promotion (pro-inflammatory) and resolution (pro-resolving) of inflammation. In addition, it is known that low-dose aspirin (acetylsalicylic acid) inhibits excessive inflammation by significantly reducing the levels of pro-inflammatory lipid mediators and increasing the synthesis of the specialized pro-resolving mediators (SPM). At the present time, however, the physiological effect of a single aspirin dose on plasma pro-resolving lipid mediator levels in healthy human subjects, or on therapeutic response in intensive care patients, is unclear. Here, we will describe the development of optimized collection, storage, extraction and liquid chromatography–tandem mass spectrometry analysis conditions for the clinical measurement of plasma lipid mediators, in response to aspirin in (i) a pilot study of healthy volunteers with the hope of extending this analysis to (ii) a randomized double blind placebo control study to evaluate the effect of aspirin on clinical outcome in intensive care patients.