Human milk (HM) is extremely important to the baby, containing a varied and balanced range of nutrients fundamental to the ideal development of the newborn. Among these nutrients, HM lipids provide 40-60% of the energy. In HM banks, the creamatocrit method is employed as a quick and solvent-free tool, however, since Folch methodology is considered reference for lipid extraction, this study compared the fatty acid (FA) composition and the lipid profile by gas chromatography with flame ionization detector (GC-FID) and direct infusion by electrospray ionization mass spectrometry (DI-ESI-MS) in pools of different HM phases, obtained from Folch and creamatocrit methods, in order to elucidate which method provides better information about HM lipids. Lipid compositon results by Folch were superior to the results obtained by creamatocrit in HM pools investigated. It means, the lipid profiles of all HM pools were more intense, and the number of FA identified by Folch was also higher in comparison to the creamatocrit method (21 and 6 FAs, respectively). GC-FID analysis in combination with principal component analysis (PCA) divided the methods, confirming a greater contribution of the Folch method. Therefore, Folch methodology revealed an effective lipid extraction in comparison to creamatocrit method.