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To target sphingolipids for diagnostic purposes, standardized methods for lipid extraction, quali\u2010 and quantitative assessments are required. In this study, HPTLC\u2010MALDI MS was adopted to establish sphingolipid and glycosphingolipid profiles in muscle, brain and serum to create a database of molecules to be searched in the preclinical and clinical investigations. Specific protocols for lipid extraction were set up based on the characteristics of the tissue or\/and fluids; this approach maximizes the HPTLC\u2010MALDI MS analytical throughput both for lipids extracted in organic and aqueous phase. This study indicates that alkaline hydrolysis is necessary for the detection of low abundant species such as Gb3Cer and ceramides in serum and Gb4Cer, CerP and HexCer in muscle tissue. The high hydrophobicity of ceramides has been overcome by the development of HPTLC plate in chloroform:methanol\/50:3.5, which increases the number and the intensity of low abundant Cer species. 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