In this study, we developed an in vivo vitiligo induction model to explore the underlying mechanisms leading to Koebner’s phenomenon and to evaluate the efficacy of therapeutic strategies. The model consisted of 12 pigmented test regions on the back of generalized vitiligo patients that were exposed to three Koebner induction methods: cryotherapy, 755 nm laser therapy, and epidermal abrasion. In addition, four cream treatments (pimecrolimus, tacrolimus, steroid and placebo) were randomly applied. Koebnerization was efficiently induced by all three induction methods. In general, cryotherapy was the best method of Koebner induction, followed by 755 nm laser therapy and epidermal abrasion. Reproducible results were obtained, which showed enhanced depigmented surface areas and higher amounts of T lymphocytes in placebo-treated test zones compared to active treated areas. Tacrolimus and local steroids were better inhibitors of Koebner’s process (P < 0.05) compared to pimecrolimus. Our in vivo vitiligo induction model is very informative to investigate vitiligo induction and to determine the efficacy of topical treatments in vitiligo. This proof of concept confirms the efficient comparison of head-to-head therapeutic strategies intra-individually in a standardized, specific and better timed way.