There is an increasing need in cosmetic clinical research for non-invasive, high content, skin imaging techniques offering the possibility on the one hand, to avoid performing invasive biopsies, and on the other hand, to supply a maximum of information on the skin state throughout a study, especially before, during and after product application. Multiphoton microscopy is one of these techniques compatible with in vivo human skin investigations, allowing human skin three-dimensional (3D) structure to be characterized with sub-µm resolution. In association with fluores-cence lifetime imaging (FLIM) and specific 3D-image processing, one can extract several quantitative parameters characterizing skin constituents in terms of morphology, density and organization. Various intracellular and extracellular constituents present specific endogenous signals enabling a non-invasive visualization of the 3D structure of epidermal and superficial dermal layers. Multiphoton FLIM applications in the cosmetic field range from knowledge to evaluation studies. Knowledge studies aim to acquire a better knowledge of skin differences appearing with aging, solar exposure or between the different skin phototypes. Evaluation studies deal with the efficacy of cosmetic anti-aging or whitening ingredients. The goal of this chapter is not to give a literature review of multiphoton FLIM applications in cosmetic clinical research, but rather to acquaint the reader with the quantitative 3D information afforded by multi-photon FLIM imaging of human skin and its interest in cosmetic clinical research.