The kinetics of permeation across epithelial and endothelial cell sheets and across cell membranes is determinant for the pharmacokinetics of a drug. In vitro transport experiments with cultured cells or artificial barriers have tremendously improved the predictability of the in vivo behaviour of tested compounds. This article focuses on the parameters and calculation methods that are used to describe permeation quantitatively, with a focus on in vitroexperiments and the prediction of intestinal absorption and blood-brain barrier passage. It shows under which in vitroexperimental conditions standard calculations are adequate and under which conditions equations should be adapted to the experimental details. The impact of volume differences between donor and receiver compartments, pH gradients, addition of albumin, accumulation in the barrier and unidirectional transport by an efflux transporter on the results is shown in simulations. The article should make researchers aware of experimental factors that affect the outcome of a permeation experiment and how to account for this during data analysis. Finally, strategies to predict the in vivo behaviour of a compound based on the in vitro data are discussed. The goal of the article is to support researchers in choosing experimental conditions and calculation methods that deliver appropriate and reproducible results in permeation studies in vitro.