<b><i>Background:</i></b> Hidradenitis suppurativa/acne inversa (HS) is a chronic, recurrent inflammatory skin disease. Its pivotal pathogenetic event is believed to be the occlusion of the hair follicle generating a perifollicular lympho-histiocytic inflammation. However, knowledge of the exact HS pathogenesis requires further research. <b><i>Objective:</i></b> To develop a human HS model applicable in preclinical research which could help to understand the pathophysiology of HS and to determine the action of therapeutic candidates. <b><i>Methods:</i></b> The 3D-SeboSkin technology was applied to maintain explants of involved and uninvolved skin of HS patients ex vivo for 3 days. Detection of differential expression of previously detected HS biomarkers was performed by immunohistochemistry in a group of female patients (<i>n</i> = 9, mean age 37.2 ± 8.4 years). <b><i>Results:</i></b> The application of the 3D-SeboSkin model preserved the structural integrity of lesional and perilesional HS skin ex vivo, as previously described for healthy skin. Moreover, the HS 3D-SeboSkin setting maintained the differential expression and pattern of several HS biomarkers (S100A9, KRT16, SERPINB3) in epidermal and dermal tissue and the appendages. <b><i>Conclusion:</i></b> We have validated HS 3D-SeboSkin as a reproducible, human model, which is appropriate for preclinical lesional and perilesional HS skin studies ex vivo.