Skin-barrier restoration following abrasive trauma is facilitated by mediator release from skin-resident cells, a process that has been investigated primarily in mice or simplified human systems with previous studies focusing on a limited number of biomarkers. Here, we demonstrate how early events caused by skin-barrier disruption can be studied in a human ex vivo skin model. Ten relevant biomarkers were recovered from the interstitial fluid by skin microdialysis with subsequent sample analysis using a multiplex platform. As a control, the biomarker profiles obtained from microdialysis sampling were compared to profiles of skin biopsy homogenates. We found that nine (GM-CSF, CXCL1/GROα, CXCL8/IL-8 CXCL10/IP-10, IL-1α, IL-6, MIF, TNF-α, and VEGF) of the 10 biomarkers were significantly upregulated in response to abrasive trauma. Only dialysate levels of CCL27/CTACK were unaffected by skin abrasion. Biomarker levels in the homogenates corresponded to dialysate levels for CCL27/CTACK, CXCL1/GROα, CXCL8/IL-8, and IL-6. However, IL-1α showed an inverse trend in response to trauma, and biopsy levels of MIF were unchanged. GM-CSF, CXCL10/IP-10, TNF-α, and VEGF were not detected in the biopsy homogenates. Our results suggest that the human ex vivo skin model is a reliable approach to study early events after disruption of the skin barrier.