<b><i>Background:</i></b> High-resolution images of the epidermis are important to understand the transdermal penetration and changes in epidermal components. Both ex vivo and in vivo technologies are available to picture the epidermal thickness (ET). So far, the illustration of the stratum corneum (SC) has not been possible without artifacts. <b><i>Objective:</i></b> Precision in vivo measurement of the ET and SC, duly considering the impact of location on the body, age, and gender. <b><i>Methods:</i></b> In this pilot study on 20 skin-healthy subjects aged 18–66 years, the ET was imaged by two-photon microscopy (2PM) and optical coherence tomography (OCT), and the SC by using 2PM at five different body sites. <b><i>Results:</i></b> On solar-exposed body areas, both the epidermis and SC are thicker compared to solar-protected areas (<i>p</i> &#x3c; 0.05), the epidermis at the gluteal region being the thickest (<i>p</i> &#x3c; 0.05). The ET decreases with age (<i>p</i> &#x3c; 0.05). Males show a thicker epidermis than females (<i>p</i> &#x3c; 0.05). <b><i>Conclusion</i></b>: 2PM provides a noninvasive method for imaging the epidermis and especially the SC in vivo<i></i> and<i></i> is optimally suited for the application of histological criteria.