Background: Atopic dermatitis is a highly heterogeneous skin disease, mainly affecting children. Introduction of biological therapies has urged the development of biomarkers to facilitate personalized therapy. Stratum corneum biomarkers emerged as a promising non-invasive alternative to skin biopsy, yet validation of spatial and biological variability is essential for their application in clinical research. Objective: To assess spatial and biological variability of stratum corneum biomarkers for atopic dermatitis. Methods: Stratum corneum was collected from 17 atopic dermatitis patients by consecutive application of eight adhesive tapes to a lesional skin site and 2 cm and 4 cm from the lesion. Two non-lesional sites at a 2 cm distance from the same lesion were collected to determine biological variability. Filaggrin degradation products (NMF) were determined by liquid chromatography and thirteen cytokines (IL-4, IL-13, IL-18, IL-31, IL-33, CCL17, CCL22, CCL27, CXCL8, IL-1α, IL-RA, IL-18, IL-22) by multiplex immunoassay. Results: Biomarker levels showed gradual changes from lesional to non-lesional skin sites at 2 cm and 4 cm; magnitude and direction of change were biomarker-specific. Intra-subject variability ranged from 17.3% (NMF) to 85.1% (CXCL8). Biomarker levels from two stratum corneum depths were highly correlated; several biomarkers showed significant depth dependence. Conclusion: Stratum corneum enables non-invasive collection of relevant immune and epidermal biomarkers, but biomarker-specific spatial and biological variability emphasizes the importance of standardized procedures for stratum corneum collection.