This study investigates lipid distributions in human skin biopsies and in vitro skin equivalents (SE) using high-resolution MALDI FTICR MSI combined with HPLC-MS/MS. SE and native skin share key spatial lipid features, particularly in the stratum corneum and epidermis. Pathway analysis further supports the biological relevance of detected lipids. These findings support the use of SE as a reproducible model for spatial lipidomics under defined conditions.