Extracellular matrix (ECM) remodeling of the skin is a continuous process necessary for maintaining tissue homeostasis. Type VI collagen (COL6) is characterized as a beaded filament, located in the dermal ECM, where COL6-α6-chain has been demonstrated upregulated in atopic dermatitis. The aim of this study was to develop and validate a competitive ELISA, targeting the N-terminal of COL6-α6-chain, named C6A6, and evaluate its associations with the dermatological condition’s atopic dermatitis, psoriasis, hidradenitis suppurativa, systemic lupus erythematosus, systemic sclerosis, urticaria, vitiligo, and cutaneous malignant melanoma in comparison, to healthy controls. A monoclonal antibody was raised and employed in an ELISA assay. The assay was developed, technically validated, and evaluated in two independent patient cohorts. Cohort 1 showed C6A6 was significantly elevated in patients with atopic dermatitis (p < 0.0001), psoriasis (p < 0.0001), hidradenitis suppurativa (p = 0.0095), systemic lupus erythematosus (p = 0.0032) and melanoma (p < 0.0001) compared to healthy donors. Cohort 2 confirmed C6A6 being upregulated in atopic dermatitis compared to healthy controls (p < 0.0001), but also associated with disease severity (SCORAD, p = 0.046) and lowered in patients receiving calcineurin inhibitors (p = 0.014). These findings are hypothesis generating, and the utility of the C6A6 biomarker for disease severity and treatment response needs to be validated in larger cohorts and longitudinal studies.