Clinical multiphoton tomography and two-photon microendoscopy provide clinicians and researchers with high-resolution in vivo optical biopsies based on two-photon autofluorescence, second harmonic generation, and fluorescence lifetime imaging. This review reflects state of the art technology and reports on applications in the fields of early stage melanoma detection, skin aging, nanoparticle imaging, tissue engineering, and in situ screening of pharmaceutical and cosmetical products. So far, more than 500 patients and volunteers in Europe, Asia, and Australia have been investigated with these novel molecular imaging tools.