Inflammation is a complex cascade process involved in the pathogenesis of a number of diseases or generated as response to external or internal stimuli. Current research is focused on the development of assays for fast identification and quantitation of inflammation biomarkers. Eicosanoids are the oxidation metabolites of polyunsaturated fatty acids (mainly 20-carbon fatty acids) that play a regulation role in inflammation and, therefore, they have proved to be involved in different pathological states such as cancer, atherosclerosis, arthritis and cardiovascular or immunological diseases. Eicosanoids can be metabolized by different oxygenase enzymes to prostanoids such as prostaglandins and thromboxanes or hydroxyl fatty acids such as hydroxyeicosatetraenoic acids and hydroxyoctadecadienoic acids. A high-throughput automated approach is here presented for direct eicosanoid analysis in biofluids such as human serum and cells culture media. The approach is based on a hyphenated system composed by a solid-phase extraction workstation (Prospekt 2 unit) on-line coupled to a liquid chromatograph-triple quadrupole-tandem mass spectrometer. The detection limits for the target analytes ranged from 0.009 to 204 pg on-column, with precision between 2.65% and 7.33%, expressed as relative standard deviation. Accuracy studies with a dual-cartridge configuration resulted in recoveries between 78.6% and 100%, which validated internally the proposed approach ensuring highly efficient cleanup of proteins and salts. The method is reliable, robust and endowed with a great potential for implementation in clinical and routine laboratories. Analysis of culture media of stem cells stimulated with arachidonic acid was carried out to evaluate its incidence on the eicosanoid profile of the exometabolome.