Background: Photodynamic therapy with Protoporphyrin IX (PpIX) is well-established for keratinocyte dysplasia. PpIX fluorescence can be quantified at the skin surface and within skin layers, but their relation is previously undescribed. The study objective was to assess the relation between skin surface PpIX fluorescence and PpIX fluorescence in epidermis and dermis. Methods: Normal buttocks skin was tape-stripped and incubated with 20 % methyl aminolevulinate cream and 20 % hexyl aminolevulinate cream under occlusion. After 3 h, skin surface PpIX fluorescence photographs were captured and 4 mm punch biopsies sampled. PpIX fluorescence microscopy was measured in regions of interest (ROI) in epidermis and superficial dermis. All PpIX fluorescence intensities were calculated in arbitrary units (AU). Results: Skin surface PpIX fluorescence intensity expressed a positive, linear relation to ROI-epidermis PpIX fluorescence (HAL- and MAL-incubated skin, p < 0.001, r² = 0.60), but not to PpIX fluorescence intensities in ROI-superficial dermis (p = 0.112). PpIX fluorescence microscopy identified higher fluorescence intensities in ROI-epidermis (median 92 AU) compared to lower fluorescence intensities in ROI-superficial dermis (median 37 AU) (p < 0.001). HAL reached higher median PpIX fluorescence intensities than MAL when measured by skin surface photographs (10230 vs. 5630 AU, p < 0.001), andbyfluorescence microscopy in ROI-epidermis (107 vs. 52.5 AU, p < 0.001) or ROI-superficial dermis (40 vs. 30.5 AU, p = 0.04). Conclusion: The positive linear relation between skin surface PpIX fluorescence photographs and epidermal PpIX fluorescence microscopy indicates that skin surface PpIX fluorescence predominantly derives from epidermis.