Interaction of nanoparticles with the skin barrier is a recent area of research that draws a lot of attention from the researchers. However, monitoring nanoparticles in or through the skin is mainly based on qualitative microscopical techniques. Yet, a quantitative approach is required for a better basic understanding. In response, a combined "multiphoton-pixel analysis" method was developed in this study for semiquantitation of gold nanoparticles penetration into different skin layers. The developed approach provides a useful tool for future studies focusing on skin penetration of nanoparticles for the aim of health risk assessment or for the design of topical and transdermal drug delivery systems.