Summary Background To date, there has been no reliable in vitro test to diagnose aspirin‐exacerbated respiratory disease (AERD). Objective To investigate potential diagnostic biomarkers for AERD using metabolomic analysis. Methods An untargeted profile of serum from asthmatics in the first cohort (group 1) comprising 45 AERD, 44 patients with aspirin‐tolerant asthma (ATA), and 28 normal controls was developed using the ultra‐high‐performance liquid chromatography (UHPLC)/Q‐ToF MS system. Metabolites that discriminate AERD from ATA were quantified in both serum and urine, which were collected before (baseline) and after the lysine‐aspirin bronchoprovocation test (Lys‐ASA BPT). The serum metabolites were validated in the second cohort (group 2) comprising 50 patients with AERD and 50 patients with ATA. Results A clear discrimination of metabolomes was found between patients with AERD and ATA. In group 1, serum levels of LTE4 and LTE4/PGF2α ratio before and after the Lys‐ASA BPT were significantly higher in patients with AERD than in patients with ATA (P < 0.05 for each), and urine baseline levels of these two metabolites were significantly higher in patients with AERD. Significant differences of serum metabolite levels between patients with AERD and ATA were replicated in group 2 (P < 0.05 for each). Moreover, serum baseline levels of LTE4 and LTE4/PGF2α ratio discriminated AERD from ATA with 70.5%/71.6% sensitivity and 41.5%/62.8% specificity, respectively (AUC = 0.649 and 0.732, respectively P < 0.001 for each). Urine baseline LTE4 levels were significantly correlated with the fall in FEV1% after the Lys‐ASA BPT in patients with AERD (P = 0.008, r = 0.463). Conclusions and Clinical Relevance Serum metabolite level of LTE4 and LTE4/PGF2α ratio was identified as potential in vitro diagnostic biomarkers for AERD using the UHPLC/Q‐ToF MS system, which were closely associated with major pathogenetic mechanisms underlying AERD.