Plant cuticle is a complex mixture of hydrophobic components that controls the uptake of pesticides by plants. Although the transport of lipophilic molecules across the cuticle has been intensively studied, development of tools to measure the cuticle polarity has received little attention. We developed a rapid and simple analytical method to evaluate the polarity of cuticles in situ. This method uses Prodan, 6-propionyl-2-(dimethylaminonaphthalene), a medium-sensitive fluorescent probe. Tests on model surfaces with varied polarity (i.e., wax paraffin, polyethylene, C18) were carried out to test the feasibility of the measurement and to optimize the application of Prodan. Moreover, on the basis of the Kamlet–Taft solvatochromic comparison method, a relationship between the emission characteristics of Prodan and the number of carbon atoms in primary alcohols mimicking the solid medium was established. After optimization, the method was validated on three natural plant cuticles (leaf of Zamiifolia, skin of green pepper, and skin of white grape).