Eicosanoids are hundreds of metabolites derived from poly‐unsaturated fatty acids (PUFAs), which regulate biological processes from multiple angles via a complex metabolic network. Targeted eicosanoid metabolomics is used to study the eicosanoid profile in biological samples but only for eicosanoids with available standards. To expand the coverage of eicosanoids detected, we identified the eicosanoids without available standards by estimation of the retention time and comparison of the MS/MS spectra with the reference ones which was collected in a database from literature. Scheduled multiple reaction monitoring‐ information dependent acquisition‐ enhanced product ion (sMRM‐IDA‐EPI) scan mode was applied in this method, which was called Spectrum Evaluation‐assisted Eicosanoid Metabolomics (SEEM). By using this method, 243 eicosanoids (167 without standards) could be relatively quantified with precision over 90 percent. We applied the method to analyze the global profile of eicosanoids secreted by human umbilical vascular endothelial cells at the basal level and with n‐3 PUFA treatment. 26 putative eicosanoids showed altered levels, despite no available standards. In general, n‐3 PUFA treatment increased most of their own metabolites and decreased the epoxy‐, hydroxyl‐ and keto‐ linoleic acid metabolites. The application of the SEEM method proved its potency of identification and quantification of eicosanoids without standards.