Widespread applications of ZnO nanoparticles (NP) in sun-blocking cosmetic products have raised safety concerns related to their potential transdermal penetration and resultant cytotoxicity. Nonlinear optical microscopy provides means for high-contrast imaging of ZnO NPs lending in vitro and in vivo assessment of the nanoparticle uptake in skin, provided their nonlinear optical properties are characterized. We report on this characterization using ZnO NP commercial product, Zinclear, mean-sized 21 nm. Two-photon action cross-section of this bandgap material (Ebg = 3.37 eV, λbg = 370 nm) measured by two techniques yielded consistent results of ηZnOσ(2ph)ZnO = 6.2 ± 0.8 μGM at 795 nm, and 32 ± 6 μGM at 770 nm per unit ZnO crystal cell, with the quantum efficiency of ηZnO = (0.9 ± 0.2) %. In order to demonstrate the quantitative imaging, nonlinear optical microscopy images of the excised human skin topically treated with Zinclear were acquired and processed using σ(2ph)ZnO and ηZnO values yielding nanoparticle concentration map in skin. Accumulations of Zinclear ZnO nanoparticles were detected only on the skin surface and in skin folds reaching concentrations of 800 NPs per μm3.