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. Multiphotonmicroscopy 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 fluorescence 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 FLIMapplications 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 multiphoton FLIM imaging of human skin and its interest in cosmetic clinical research.