Sensitisation leading to food allergy may occur through the skin. Quantitative data on epidermal allergen penetration as a prerequisite for this remain limited. This study quantifies epidermal penetration of gluten and hydrolysed wheat protein (HWP) in 18 patients with challenge-confirmed WALDA (wheat allergy dependent on augmentation factors) and 12 healthy controls (HC). After 1 h of epicutaneous wheat application, 20 consecutive tape strips (TS) were collected, and wheat protein concentration was quantified by gliadin-specific ELISA. Skin barrier status was assessed by electrical impedance spectroscopy (EIS). Serum gliadin levels were measured before and 90 min after wheat application. Gliadin was detected across all TS layers for gluten and HWP. Penetration levels did not differ between patients and controls. Skin barrier status assessed by EIS did not differ significantly between individuals with and without a history of atopic dermatitis (p = 0.27). No correlation was observed between EIS-assessed skin barrier status and gliadin penetration. Serum gliadin was not increased after epicutaneous wheat application. This study using TS demonstrates for the first time that wheat allergens penetrated into the epidermis but could not be detected in the serum. Neither wheat allergy nor skin barrier status was associated with increased stratum corneum penetration. These findings suggest that epidermal uptake alone may not be sufficient to explain sensitisation.