Dry skin in atopic dermatitis (AD) mainly results from barrier impairment due to deficiency of ceramide and natural moisturizing factors including pyrrolidone carboxylic acid (PCA) in stratum corneum (SC). Caspase-14 cleaves filaggrin monomers to free amino acids and their derivatives such as PCA, contributing natural moisturizing factors. Cytokines in the corneocytes represent cutaneous inflammation severity of AD patients.To analyze the correlations of PCA, caspase-14 and cytokines in corneocytes with clinical severity, barrier function and skin inflammation, those were quantitated.A total of 73 persons were enrolled: 21 patients with mild AD, 21 with moderate-to-severe AD, 13 with X-linked ichthyosis (XLI) as a negative control for filaggrin gene (FLG) mutation, and 18 healthy controls. Skin barrier functions such as basal transepidermal water loss (TEWL), stratum corneum (SC) hydration and skin surface pH were measured. To collect corneocytes, stripping with D-squame discs was done on lesional and non-lesional skin. And then PCA was isolated from D-squame discs and quantitated by LC-MS/MS. Cytokine assays were performed.The quantity of PCA and caspase-14 was decreased in inflammatory lesions compared to non-lesion in AD patients. And the amounts of PCA and caspase-14 in the lesion of AD patients correlated with clinical severity as determined by eczema area and severity index score and the skin barrier functions. Also, the expressions of TNF-α and IL-13 inversely correlated with PCA quantity.The quantity of PCA or caspase-14 in the corneocytes of the lesional skin of AD patients reflects the clinical severity, skin barrier function and the degree of lesional inflammation.