The skin is a highly accessible organ and thus provides an attractive immune environment for cost-effective, simple and needle-free delivery of vaccines and immunomodulators. In this study, we pretreated mouse skin with an anionic surfactant, sodium lauryl sulfate (SLS), for a short period of time (10 min) followed by epicutaneous vaccination with hen egg lysozyme (HEL) antigen. We demonstrated for the first time that pretreatment of skin with surfactant SLS significantly enhances the production of antibody to HEL. Short-term pretreatment with SLS disorganized the stratum corneum, extracted partial lamellar lipids, induced the maturation of Langerhans cells and did not result in epidermis thickening. To reveal the mechanism underlying these changes, particularly at the molecular level, we employed a novel proteomics technique, using ultrafiltration capillaries and mass spectrometry to identify in vivo proteins/peptides secreted in the SLS pretreated skin. Two secretory proteins, named as calcium binding protein S100A9 and thymosin ß4, were identified by this novel technique. These two proteins thus may provide new insight into the enhancing effect of surfactants on skin vaccination.