Atopic dermatitis (AD) has a complicated pathogenesis and its clinical features vary greatly among patients. Although many clinical parameters have been reported, it remains difficult to evaluate AD skin conditions adequately. To support better evaluation of AD patients, we attempted to develop a new, objective and noninvasive method that assesses skin condition in AD using biochemical markers in the skin’s horny layer (HL). Thirty-six patients with AD, 8 with psoriasis and 16 healthy volunteers were recruited. HL samples were obtained by tape stripping from involved and uninvolved skin of the forearms. Expression levels of 6 proteins in the HL [fatty acid-binding protein-5 (FABP-5), squamous cell carcinoma antigens 2 (SCCA2), α-enolase, annexin II, apolipoprotein AI and albumin] were analyzed by immunoblotting and compared with clinical data. The 6 proteins were detected at a high level in AD skin lesions, but scarcely in the normal controls. FABP-5 showed correlation with the local severity of the involved skin. Annexin II, apoprotein AI and albumin showed correlation with the severity of specific eruptions. SCCA2 correlated significantly with total serum IgE level. Albumin levels in the uninvolved skin of AD patients showed significant correlation with the local severity in the involved skin of the same patient and with the trans-epidermal water loss. Albumin levels in psoriatic skin were very low, even with scratch marks, compared to those in AD skin. FABP-5, albumin and some other proteins in HL seem to be useful as biomarkers to evaluate inflammation and skin barrier conditions in AD patients.