Numerous studies are underway to identify an effective dynamic therapy against cutaneous melanoma. Dynamic therapy is based on the interaction of an external energy source and a molecule sensitive to this energy, which triggers a series of reactions resulting in tumor cell death. The adequate distribution and concentration of the sensitizer in tumor cells can lead to obtaining the expected anticancer effects. Protoporphyrin IX (PpIX) is a well-known endogenous sensitizer. Its accumulation in target cells can reach photosensitizing concentrations by the exogenous application of its precursor, 5-aminolevulinic acid (ALA). This study aimed to find an effective protocol for ALA delivery that induces a higher sensitizing PpIX deposition in murine cutaneous melanoma tumors. For this purpose, melanoma-bearing mice were subjected to five different protocols of ALA delivery: topical administration MAL cream (20% w/w methyl aminolevulinate hydrochloride, incubation time: 12h), intratumoral ALA injection (dose: 200 mg/Kg b.w., incubation time: 2 h), and intraperitoneal ALA injection (dose: 200 mg/Kg b.w., incubation time: 3, 4, 5 h). The amount of PpIX extracted from the melanoma tumors (B16F10) was compared with that extracted from a non-melanoma tumor model (A431) where the PpIX accumulation was induced by a clinical protocol of ALA delivery commonly applied for photodynamic therapy. The detection and quantification of PpIX were carried out by applying Steady-state fluorescence spectroscopy (SSFS) by in vivo and in vitro measurements, with the latter involving a chemical extraction method. In vitro measurements showed that the PpIX concentration extracted from the melanoma tumor was greater by intraperitoneal (3 h) (0.31±0.05 µM) and intratumoral (0.39±0.17 µM) protocols. Through the topical protocol, it was possible to avoid the healthy region sensitization, and the concentration of PpIX extracted from the melanoma tumor (0.15±0.04 µM) was almost the same amount as that generated by the clinical protocol (0.18±0.05 µM). The results suggested that a photosensitive amount of PpIX can be synthesized within the melanoma tumor by the intratumoral, intraperitoneal (3 h) protocols.