Penetration profiles of topically applied drugs and cosmetic products provide important information on their efficacy. The application of tape stripping in combination with UV/VIS spectroscopy is checked to determine the local position of topically applied substances inside the stratum corneum, the penetration profile. The amount of corneocytes removed with each tape strip is quantified via the particle-dependent absorption, the pseudoabsorption, in the visible spectral range. The concentration of a typical UV filter substance, 4-methylbenzylidene camphor, is determined by optical spectroscopy using the tape strips removed originally. In this case, a time-dependent increase in the absorbance must be taken into account. Laser scanning microscopic investigations confirm that the nonhomogeneous distribution of the filter substance, on the strips, can explain this spectroscopic behavior. When reaching a homogeneous distribution, the UV spectroscopic signal reflects the correct concentration. These spectroscopic values are compared with high performance liquid chromatography (HPLC) data. The values obtained with both methods for the concentrations of 4-methylbenzylidene camphor are in good agreement. The data obtained are used to illustrate the determination of a penetration profile of a UV filter substance. The results demonstrate that the described protocol is well suited to characterize, in a simple manner, topically applied substances that have a characteristic UV/VIS absorption band.