The lipids of the stratum corneum comprise the most important components of the skin barrier. In patients with ichthyoses or atopic dermatitis, the composition of the skin barrier lipids is disturbed resulting in dry, scaly, itching erythematous skin. Using the latest X-Ray Photoelectron Spectroscopy (XPS) technology, we investigated the physiological skin lipid composition of human skin and the effects of cold atmospheric plasma treatment on the lipid composition. Skin lipids were stripped off forearms of six healthy volunteers using the cyanoacrylate glue technique, plasma treated or not and then subjected to detailed XPS analysis. We found that the human lipid skin barrier consisted of 84.4% carbon (+1.3 SEM%), 10.8% oxygen (+1.0 SEM%) and 4.8% nitrogen (+0.3 SEM%). The composition of physiological skin lipids was not different in males and females. Plasma treatment resulted in significant changes in skin barrier lipid stoichiometry. The total carbon amount was reduced to 76.7%, and the oxygen amount increased to 16.5%. There was also a slight increase in nitrogen to 6.8%. These changes could be attributed to reduced C-C bonds and increased C-O, C=O, C-N and N-C-O bonds. The moderate increase in nitrogen was caused by an increase in C-N and N-C-O bonds. Our results show for the first time that plasma treatment leads to considerable changes in the human skin lipid barrier. Our proof of principle investigations established the technical means to analyse, if plasma-induced skin lipid barrier changes may be beneficial in the treatment of ichthyotic or eczematous skin.