The increased ability of TMX-202 (derivative of imiquimod) to penetrate the intact stratum corneum (SC) and the follicular orifices of porcine ear skin was shown ex vivo using confocal Raman microscopy and laser scanning microscopy. Moreover, to assess whether TMX-202 is able to reach the immune cells, Langerhans cells extracted from pretreated human skin were investigated ex vivo using confocal Raman microscopy combined with multivariate statistical methods. Tracking the Raman peak of dimethyl sulfoxide centered at 690 cm(−1), the absorption of TMX-202 containing formulation by Langerhans cells was shown. To answer the question whether the TMX-202 active ingredient is able to reach Langerhans cells, the attraction of immune cells to TMX-202 containing formulation treated skin was measured in the in vivo rodent model Mastomys coucha. The results show that TMX-202 active ingredient is able to reach Langerhans cells after penetrating through the intact skin and subsequently attract immune cells. Both the intercellular/transcellular as well as the follicular pathways allow the penetration through the intact barrier of the SC.