Extraction and fractioning of microalgal lipids have been recurrently studied because of the market value of certain lipid families (i.e. carotenoids and xanthophylls) and the opportunity that microalgal oils represent for obtaining biofuels. Most of the methods developed for the total extraction of microalgal lipids require a subsequent separation for the different families (i.e neutral lipids, polar lipids) to be quantified. Moreover, most of them use toxic and expensive solvents. In the present study less toxic solvents that are widely used in the industry, such as hexane, acetone and, ethanol were tested in different orders for the extraction of microalgal biomass of different lipid composition and content (i.e. three different levels of nutritional stress). A comparison of the methods proposed by Bligh and Dyer, Folch and, Hara and Radin is also presented. The new method achieves microalgal total lipid extraction and separation of the lipid families simultaneously.