Zinc oxide nanoparticles (ZnO NPs) are commonly used as UV filters in commercial sunscreen products. Their penetration into the skin is intensively discussed in the literature. In the present in vivo study, penetration of ZnO NPs (30 nm in size) into human skin was investigated by multiphoton tomography. Based on the non-linear effects of a second harmonic generation and hyper-Rayleigh scattering, the distribution of ZnO NPs in the horny layers of the epidermis, as well as the furrows, wrinkles and orifice of the hair follicles was analyzed. This method permitted distinguishing between the particulate and dissolved forms of Zn. A detection limit of 0.08 fg/µm³ was estimated. Taking advantage of this sensitivity, it was clearly shown that ZnO NPs penetrate only into the outermost layers of stratum corneum, furrows and into the orifices of the hair follicles and do not reach the viable epidermis.