Photodynamic therapy (PDT) with 5-aminolevulinic acid (ALA) or its derivatives as precursors of protoporphyrin IX (PPIX) is routinely used in dermatology for the treatment of various pathologies. However, this methodology suffers to some extent from a limited efficacy. Therefore, the main goal of this study was to investigate the modulation and pharmacokinetics of PPIX buildup after a 5 h incubation with ALA (1.5 mM) and one of its derivatives, the hexyl ester of ALA (h-ALA) (1.5 mM), on the human epidermal equivalent Epidex™. PPIX production was modulated with (L ) ascorbic acid iron (II) salt (LAI) or the iron (II)-specific chelating agent deferoxamine (DFO). PPIX fluorescence from the Epidex™ layers was measured up to 150 h after the precursor administration using a microspectrofluorometer (λ_ex: 400 ± 20 nm; λ_det: 635 nm). The maximum PPIX fluorescence intensity induced by h-ALA was about 1.7× larger than that induced by ALA. The addition of DFO resulted in a more than 50% increase in PPIX fluorescence for both precursors. The decay half life measured for PPIX fluorescence is 30 and 42.5 h, respectively, for ALA and h-ALA. These half lives are doubled when the samples contain DFO. In the samples with the highest fluorescence intensity, a modified fluorescence spectrum was observed after 10 h, with the emergence of a peak at 590 nm, which is attributed to zinc protoporphyrin IX (Zn PPIX).