The current study focused on the development of an ex vivo human skin model to investigate the added value of nanoparticle-based drug delivery systems on skin penetration and drug-mediated efficacy compared to conventional drug formulations for the topical therapy of inflammatory skin diseases. In order to induce different types and intensities of skin barrier disruption, physical and chemical means were applied on human skin explants obtained from plastic surgery. Comparative studies on structural integrity, biophysical parameters and cytokine levels were performed three times within 48 hours in two culture systems of skin. Cultures of skin maintained in tissue media preserved key skin barrier parameters and viability better than medium-free cultures in humidified chambers. The standardization of 50-times tape stripping and 4-hour sodium lauryl sulfate (5% w/v) pretreatment was reliable: transepidermal water loss values and interleukin-6 /-8 levels as examples of a wide range of affected inflammatory mediators were increased reproducibly compared to intact skin. Different structural and biological changes were induced, which are usually associated with pathological changes in diseased skin. Transepidermal water loss measurements provide a noninvasive screening tool to control and standardize skin barrier disruption ex vivo. Based on these results, time- and tissue-sparing protocols and methods for further investigations were adapted to a short-term skin culture system. Intradermal microdialysis in one experimental setup including up to nine sets for up to 24 hours enabled comparison of intact versus physically and chemically barrier-disrupted skin on the skin of one donor as well as three dexamethasone formulations (ethyl cellulose nanocarriers, nanocrystals and a conventional cream) in parallel. The application of highly sensitive detection methods, such as liquid-chromatography-tandem-mass spectrometry and Luminex R multiplex technology, provided a complex set of data on skin penetration and biological effects. The model successfully demonstrated that skin barrier disruption and the characteristic properties of the dexamethasone formulations affected drug penetration differently. Penetration rates in chemically treated skin were lower compared to tape-stripped skin. Nanocrystals quickly and effectively penetrated intact and barrier-disrupted skin. Thus, significantly higher dermal drug concentrations were achieved within six hours compared to the other formulations. The benefit of encapsulation in ethyl cellulose nanocarriers was more pronounced in intact skin. The results were largely in line with the prediction made based on in vitro release kinetics and Franz diffusion cell studies. High local cytokine levels indicated a trauma induced by probe insertion, which restricted the estimation of drug-mediated efficacy over time. Nevertheless, evidence was found that the application of nanocrystals was associated with high dermal cytokine levels, although significantly higher amounts of the anti-inflammatory drug penetrated the skin. This could point toward an irritative potential of the nanocrystals. In summary, conclusion can be drawn about drug penetration and local biological effects in intact versus barrier-disrupted skin after the application of up to three different drug formulations. Such a complex screening tool can help to characterize the added value of each individual nanoparticle-based drug delivery systems compared to conventional formulations for different pathological skin changes in order to determine the most appropriate for a particular clinical indication. To answer the questions as to whether and to which extent nanoparticles are capable of translocating across an intact skin barrier, an in situ imaging mode based on wide-field two-photon microscopy was implemented. The analysis of full-thickness samples allowed sample preparation steps to be reduced to a minimum, which mainly avoids the production of artifacts. Moreover, both interfollicular and follicular penetration were investigated in histomorphological correlation. Proof-of-concept work on fluorescently tagged nanoparticles revealed that the vast majority of nanoparticles remained in the upper Stratum corneum. However, rare events of deeper penetration were also observed in intact skin close to regions with high focal nanoparticle aggregations in the Stratum corneum and in the infundibulum of hair follicles. Total internal reflection fluorescence microscopy confirmed barrier crossing with high sensitivity. Individual nanoparticles as well as clusters of nanoparticles were detected in the Stratum corneum and within the epidermal layer directly beneath the Stratum corneum. The combination of both technologies provides highly important insights into penetration processes and pathways as well as into the barrier function of the skin, which are crucial for the development of nanoparticle-based drug delivery systems for improved topical application of drugs for inflammatory skin diseases.