Skin equivalents (SE) are widely used as reproducible in vitro models of human skin (HS), yet detailed spatial comparisons of lipid organization between engineered and native tissue remain limited. Because lipids are central to epidermal barrier function, hydration, and signaling, their spatial distribution can be used to assess model fidelity. Here, we map lipid localization in HS and SE by high-resolution mass spectrometry imaging and relate sphingolipid intermediates to the expression of selected converting enzymes across keratinocyte differentiation states.