Two-photon laser scanning fluorescence microscopy (TPM) has been shown to be advantageous for imaging optically turbid media such as human skin. The ability of performing three-dimensional imaging without presectioning of the samples makes the technique not only suitable for noninvasive diagnostics but also for studies of topical delivery of xenobiotics. Here, TPM is used as a method to visualize both autofluorescent and exogenous fluorophores in skin. Samples exposed to sulforhodamine B have been scanned from two directions to investigate attenuation effects. It is shown that optical effects play a major role. Thus, TPM is excellent for visualizing the localization and distribution of fluorophores in human skin, although quantification might be difficult. Furthermore, an image-analysis algorithm has been implemented to facilitate interpretation of TPM images of autofluorescent features of nonmelanoma skin cancer obtained ex vivo. The algorithm was designed to detect cell nuclei and currently has a sensitivity and specificity of 82% and 78% to single cell nuclei. However, in order to detect multinucleated cells, the algorithm needs further development.