Transepidermal water loss (TEWL) is the most widely used objective measurement for assessing the barrier function of skin in healthy individuals but also patients with skin diseases that are associated with skin barrier dysfunction, such as atopic dermatitis. TEWL is the quantity of condensed water that diffuses across a fixed area of stratum corneum to the skin surface per unit time. The water evaporating from the skin is measured using a probe that is placed in contact with the skin surface and contains sensors that detect changes in water vapor density. TEWL can be measured using an open-chamber, unventilated-chamber, or condenser-chamber device. It is a sensitive measure that is affected by properties of the surrounding microclimate such as environmental humidity, temperature, and airflow and should be measured under controlled conditions. TEWL varies significantly across different anatomical sites and also depends on sweat gland activity, skin temperature, and corneocyte properties. Here we describe how to optimally use TEWL measurements as a skin research tool in vivo and in vitro.