In order to prepare for a validation study to compare percutaneous absorption through reconstructed human epidermis with ex vivo skin absorption through human and animal skin, nine test compounds, covering a wide range of physicochemical properties were selected, namely: benzoic acid; caffeine; clotrimazole; digoxin; flufenamic acid; ivermectin; mannitol; nicotine; and testosterone. The donor and receptor media for the test substances, the addition of a solubiliser for the lipophilic compounds, as well as the stability and solubility of the test substances in the vehicles, were systematically analysed. Hydrophilic molecules, being freely soluble in water, were applied in buffered saline solutions. In order to overcome solubility restrictions for lipophilic compounds, the non-ionic surfactant, Igepal CA-630, was added to the donor vehicle, and, in the case of clotrimazole and ivermectin, also to the receptor fluid. The model molecules showed a suitable solubility and stability in the selected donor and receptor media throughout the whole duration of the test.