Aims: Follicular drug delivery is the prerequisite for an effective treatment of androgenetic alopecia or other reasons of premature hair loss. Methods: The follicular penetration of caffeine, applied topically in a shampoo formulation for 2 min, was measured with highly sensitive surface ionization in combination with mass spectroscopy, a selective method for the detection of very small quantities of transcutaneously absorbed substances in the blood. An experimental protocol, developed to selectively block the follicular pathway within the test area, was used. Based on this principle, a clear distinction between interfollicular and follicular penetration of topically applied caffeine was feasible. Results: After 2 min, caffeine penetrated via the hair follicles and stratum corneum. Conclusion: It was found that the penetration via hair follicles was faster and higher compared with the interfollicular route and that hair follicles are the only pathway for fast caffeine absorption during the first 20 min after application.