Atopic dermatitis (AD) is a common childhood chronic inflammatory skin disorder that can significantly impact quality of life and has been linked to the subsequent development of food allergy, asthma, and allergic rhinitis, an association known as the “atopic march. The aim of this study was to identify biomarkers collected non-invasively from the skin surface in order to predict AD before diagnosis across a broad age range of children. Non-invasive skin surface measures and biomarkers were collected from 160 children (3–48 months of age) of three groups: (A) healthy with no family history of allergic disease, (B) healthy with family history of allergic disease, and © diagnosed AD. Eleven of 101 children in group B reported AD diagnosis in the subsequent 12 months following the measurements. The children who developed AD had increased skin immune markers before disease onset, compared to those who did not develop AD in the same group and to the control group. In those enrolled with AD, lesional skin was characterized by increased concentrations of certain immune markers and transepidermal water loss, and decreased skin surface hydration. Defining risk susceptibility before onset of AD through non-invasive methods may help identify children who may benefit from early preventative interventions.