The transfollicular administration of pharmacologically active molecules is of current therapeutic interest, mainly with regard to delivery to specific sites of the hair follicle (HF) and the reduction of hepatic metabolism and systemic toxicity. HF are privileged pathways for specific molecules depending on formulations, which enter faster into these shunts than through the stratum corneum. The aim was to optimize the delivery of fluorescent microspheres into the HF, thereby, developing a standardized protocol for follicular targeting with microspheres. The number of HF showing penetration, as well as the depth of penetration, was determined. Freshly excised skin samples with terminal HF were divided into groups, with or without prior treatment with cyanoacrylate skin surface stripping-technique (CSSS). Thereafter microspheres at a size of 0.75-6.0 microm were applied according to the developed standardized protocol. Skin biopsies were obtained, shock-frozen, and sectioned in 5 microm slices. We demonstrated a selective penetration route of the microspheres into the HF. Optimal microsphere size proved to be approximately 1.5 microm, with a 55% rate of all HF, and with a maximum penetration depth of >2300 microm. Without previous CSSS treatment of the skin, the transfollicular microsphere penetration was below 27% with a maximum penetration depth of 1000 microm. Thus, the basis for follicular targeting of essential structures containing stem cells for keratinocytes, melanocytes, and mast cells has been laid.