Decreasing incubation time, as well as enhanced PpIX production, are present challenges for topical photodynamic therapy (PDT). There are reports concerning the existence of a strong relationship between site temperature and porphyrin synthesis in biological tissue, that suggest temperature increase in the tissue can improve the formation of PpIX. The main objective of this study is to determine whether the temperature change of the tissue favors the production of PpIX. Creams containing aminolevulinic acid (ALA) and methyl aminolevulinate (MAL) were topically administered for 30 min on healthy skin of rats' back and the formation of PpIX was evaluated for 180 min. The animals were divided into 5 groups: cooling tissue to 20 °C or heating tissue to 40 °C (either before or after incubation of the cream) and control group (unchanged temperature). The tissue temperature was evaluated by thermography. The influence of temperature was evaluated both concerning cream penetration and the production of PpIX. It was found that both ALA and MAL led to an increase of about 20% PpIX production when the tissue was warmed before incubation of the cream, suggesting that the penetration improved. When the thermal change was promoted after incubation of the cream, the production of PpIX decreased both by heating and cooling, probably related to enzyme modification. The results found in this study suggest that the increase of tissue temperature before the cream incubation can improve the clinical protocols of topical PDT using ALA or MAL, improving the efficiency of the procedure by increasing the production of PpIX and allowing the decrease of the incubation period.