This study presents a novel non-lethal sampling method for assessing fatty acid (FA) composition in juvenile European sea bass (Dicentrarchus labrax) using subcutaneous white muscle biopsies. This research aimed to evaluate the suitability of the biopsy for FA analysis using two lipid extraction protocols and comparing them to a lethal routine method. The results showed that a mass of fresh tissue as low as 1.4 mg provided good quality FA chromatograms for both reserve and membrane lipids. Although the biopsy method displayed high variability in terms of FA quantity among intra-individual replicates, it showed good FA profile repeatability in both reserve and membrane lipids. The study highlights the potential of this non-lethal approach for studying FA dynamics in fish, with its application being particularly promising for ecological and experimental studies. However, careful biopsy implementation is recommended to account for potential lipid droplet and lipid distribution variability within the tissue.