Abstract There is a constant need for simple, economical and time-efficient methods which allow evaluating a compound's ability to penetrate the biological membrane, one of the key parameters needed to characterize biologically active compounds. In the paper we propose a new method of permeability determination. Instead of detecting the compound's concentration directly, we employ an approach in which the membrane interface is labeled with a fluorescein lipid probe; the probe is sensitive to the presence of charged compounds. The fluorescence intensity changes of the dye permanently attached to both sides of a model lipid bilayer are measured. Specifically, the time course of the fluorescence intensity changes following a rapid induction of a non-equilibrium state of the sample allows the evaluation of the membrane permeability for the compound. The method was validated by the determination of the phenyltin compound's transport through the model phosphatidylcholine unilamellar liposome bilayer.