The skin deploys multiple barriers to protect internal tissues and organs, as well as the skin itself, from the external environment. Each of the skin constituents—epidermis, dermis, sebaceous glands, sweat glands, hair, and adipose tissue—contributes to making these barriers competent to maintain proper biological functions. Among these constituents, the epidermis (the outermost layer of skin) is the first line of defense, so it is most responsible for skin barrier functions. A competent skin barrier is needed to protect against excess loss of water and other endogenous compounds from body, oxidative stress, sun and other sources of light irradiation, thermal stress, microbial invasion, substance permeation, and mechanical stress. In this chapter, we describe methods to assess both the epidermal permeability barrier and the light irradiation barrier using in vitro and ex vivo skin organ-cultured systems.