3 D human epidermal equivalents (HEEs) are a state-of-the-art organotypic culture model in pre-clinical investigative dermatology and regulatory toxicology. Here, we investigated the utility of electrical impedance spectroscopy (EIS) for non-invasive measurement of HEE epidermal barrier function. Our setup comprised a custom-made lid fit with 12 electrode pairs aligned on the standard 24-transwell cell culture system. Serial EIS measurements for seven consecutive days did not impact epidermal morphology and readouts showed comparable trends to HEEs measured only once. We determined two frequency ranges in the resulting impedance spectra: a lower frequency range termed EIS correlated with keratinocyte terminal differentiation independent of epidermal thickness and a higher frequency range termed EIS correlated with thickness. HEEs generated from CRISPR/Cas9 engineered keratinocytes that lack key differentiation genes , or confirmed that keratinocyte terminal differentiation is the major parameter defining EIS. Exposure to pro-inflammatory psoriasis- or atopic dermatitis-associated cytokine cocktails lowered the expression of keratinocyte differentiation markers and reduced EIS. This cytokine-associated decrease in EIS was normalized after stimulation with therapeutic molecules. In conclusion, EIS provides a non-invasive system to consecutively and quantitatively assess HEE barrier function and to sensitively and objectively measure barrier development, defects and repair.