A new a microwave assisted extraction method (MAE) for fish lipids was both optimized by means of a central composite design and validated using a certified reference fish (1946, NIST). The innovation of this work was to verify the stability of the extracted fish lipids after MAE. Lipid content of the certified material obtained by MAE (10.07 ± 0.25 g/100g) was similar to the value declared in the certificate (10.17 ± 0.48 g/100g). The effect of microwave on MAE did not alter the fatty acid composition of fish and did not form lipid oxidation compounds at higher levels than the conventional extraction. Scanning electron microscopy showed that MAE causes total fish tissue disruption, thus, lipid migration to the solvent is more efficient and faster in comparison to the conventional method. The new method was applied to fish samples with different lipid contents (tilapia, pacu, hake and tuna). No significant differences were found between MAE and the conventional method regarding total lipids and fatty acid contents. MAE can be applied to fish lipid determination since the results showed good precision and accuracy. In addition, it is a fast and robust technique with low solvent consumption when compared to the conventional extraction.