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The antioxidant system of the human body plays a crucial role in maintaining redox homeostasis and has an important protective function. Carotenoids have pronounced antioxidant properties in the neutralization of free radicals. In human skin, carotenoids have a high concentration in the stratum corneum (SC)—the horny outermost layer of the epidermis, where they accumulate within lipid lamellae. Resonance Raman spectroscopy and diffuse reflectance spectroscopy are optical methods that are used to non-invasively determine the carotenoid concentration in the human SC in vivo. It was shown by electron paramagnetic resonance spectroscopy that carotenoids support the entire antioxidant status of the human SC in vivo by neutralizing free radicals and thus, counteracting the development of oxidative stress. This review is devoted to assembling the kinetics of the carotenoids in the human SC in vivo using non-invasive optical and spectroscopic methods. Factors contributing to the changes of the carotenoid concentration in the human SC and their influence on the antioxidant status of the SC in vivo are summarized. The effect of chemotherapy on the carotenoid concentration of the SC in cancer patients is presented. A potential antioxidant-based pathomechanism of chemotherapy-induced hand-foot syndrome and a method to reduce its frequency and severity are discussed.