Background Noninvasive diagnostic techniques allow for morphological and morphometric in vivo evaluation of the skin. Optical coherence tomography (OCT) is a method that allows visualization of dermal structures up to several 100 μm with a resolution of 3–7.5 μm. Objectives The aim of the study was the morphological and morphometric assessment of rats' skin using SD-OCT. Animals Fifteen male Wistar rats, aged 3 and 8 months, weighing 350–450 g. Materials and Methods The skin of the plantar metatarsal area of the right pelvic limb was assessed. The measurements were performed using Spectral Domain Optical Coherence Tomography (SD-OCT) scans and histological images. Results Spectral Domain Optical Coherence Tomography could determine the boundary between the epidermis and the dermis. Apart from the stratum corneum (SC), it did not allow for the differentiation of the individual epidermal layers. In the SD-OCT and in the histological examination, the mean thicknesses of the layers (μm ± SD) were (respectively): SC 33.053 ± μm(SD 5.85, 29.675 ± 5.54; epidermis 88.2 ± 7.97, 65.126 ± 13.23; dermis 259.86 ± 18.29, 166.05 ± 31.88 μm. There was a correlation between the total epidermal SD-OCT and histological measurements (r = 0.43, p = 0.05). Bland–Altman plots revealed a bias of −19.18 (95% confidence interval) –39.21 to 0.84 μm) in the case of live epidermis (stratum granulosum, stratum spinous, stratum basale). Conclusions and clinical relevance Spectral Domain Optical Coherence Tomography can be used to evaluate rat epidermis and dermis. The method enables the differentiation of the SC, as well as the epidermis and the dermis. SD-OCT and histological thickness dimensions of the epidermis and skin differ.