BACKGROUND Chronic endobronchial inflammation is a hallmark of pediatric asthma and involves the arachidonic acid pathway. Its non-volatile metabolites can be quantified in the exhaled breath condensate (EBC), and single substances have been studied as non-invasive biomarkers for the diagnosis and monitoring of children with asthma. The aim of this study was to compare the content and profile of a wider range of eicosanoids in the EBC between patients and a control group. METHODS EBC was sampled from 33 children (aged 12.4 ± 3.1 years) with stable atopic asthma (26 on inhaled steroid treatment) and 25 healthy controls (11.8 ± 3.2 years). Validated high performance liquid chromatography coupled with a tandem mass spectrometry platform (HPLC-MS2 ) was used to measure 13 different compounds. In addition, exhaled nitric oxide levels (FeNO) were measured and bronchial hyperresponsiveness (BHR) was assessed by an exercise challenge test in all subjects. An analytical approach was used for multivariate regression modeling of disease status using the most relevant variables. RESULTS The levels of PGEM (P < 0.001), PGD2 (P < 0.001), 6keto-PGF1α (P = 0.03), LTC4 (P < 0.001), trans-LTC4 (P = 0.04), and 5HETE (P = 0.02) were significantly higher in asthmatics compared to healthy children, while 11-dehydro TXB2 was significantly less abundant (P = 0.02). The eicosanoids asthma classification ratio (EACR) was computed as the logistic regression function using four variables: PGEM, PGD2, LTC4, and 5HETE. This composite parameter discriminated asthmatic from healthy children better than FEV1, FeNO, or BHR. CONCLUSIONS Complementary measurements of PGEM, PGD2, LTC4, and 5HETE in small-volume EBC samples are feasible by HPLC-MS2 and showed a specific profile in our study population. EACR should be evaluated further in the context of diagnosing and monitoring childhood asthma.