Presently, percutaneous absorption of potentially hazardous chemicals in humans can only be assessed in animal experiments, in vitro, or predicted mathematically. Our aim was to demonstrate the proof-of-principle of a novel quantitative in vivo assay for percutaneous absorption: confocal Raman micro-spectroscopy (CRS). The advantages and limitations of CRS for health risk assessments are discussed. 2-butoxyethanol, toluene, and pyrene were applied in pure form, diluted in water, or in ethanol on the skin of three healthy volunteers. CRS measurements were done following application for 15 min and 3 hours. The concentrations of the three substances as a function of distance to the skin surface were calculated and further analyzed with regard to mass transport into the stratum corneum (μg/cm(2)) and the flux through the stratum corneum (μg/cm(2)h). The results were compared with the available data from literature. Considering the preliminary nature of these data, good accordance with data from the literature was observed. In addition, we observed that 2-butoxyethanol penetrates markedly faster when dissolved in water as compared to ethanol. This observation is also in agreement with previous results. CRS has the potential to provide fast, accurate and reliable results for advanced studies of in vivo percutaneous absorption kinetics of hazardous substances in human skin. This will require further research with other substances and under differing conditions.