The validation of the molecular networking of NEO-PUFAs was accomplished through four distinct strategies. Firstly, the topology of the molecular network generated using the NEO-MSMS standards as input was examined to determine if the uploaded MS/MS data could highlight structural similarities among the standards present in the database. Next, we generated four feature-based molecular networks for each PUFAs, encompassing all types of oxidation. Additionally, a feature-based network was established, including all four fatty acids, to identify potential compounds based on observed connections within the molecular network from which was created our putative library. Moreover, the examination of oxPUFA data played a crucial role in identifying specific putative compounds which were subsequently added to the putative library. Finally, the dereplication efficiency of the NEO-MSMS oxylipins database implemented in GNPS libraries was estimated by annotating the molecular network obtained from blood serum and microalgae extract, and the oxPUFA data large oxylipins/NEO-PUFAs coverage was revealed by highlighting related oxylipins.