A comparative study of three detergent-free protein extraction protocols—a differential centrifugal fractionation, a delipidation protocol based on the Bligh and Dyer method, and the trifluoroethanol addition as cosolvent to an aqueous buffer—was performed on white adipose tissue. The performance of the protocols directly compatible with liquid chromatography–electrospray ionization–mass spectrometry (LC–ESI–MS) was evaluated based on the total protein extraction yield and the protein recovery from different functional and cellular compartments. The most suitable method for the extraction of white adipose tissue proteins from a wide range of cellular and structural compartments was the delipidation protocol based on the Bligh and Dyer method.