Over the years, in vitro Franz diffusion experiments have evolved into one of the most important methods for researching transdermal drug administration. Unfortunately, this type of testing often yields permeation data that suffer from poor reproducibility. Moreover, this feature frequently occurs when synthetic membranes are used as barriers, in which case biological tissue-associated variability has been removed as an artefact of total variation. The objective of the current study was to evaluate the influence of a full-validation protocol on the performance of a tailor-made array of Franz diffusion cells (GlaxoSmithKline, Harlow, UK) available in our laboratory. To this end, ibuprofen was used as a model hydrophobic drug while synthetic membranes were used as barriers. The parameters investigated included Franz cell dimensions, stirring conditions, membrane type, membrane treatment, temperature regulation and sampling frequency. It was determined that validation dramatically reduced derived data variability as the coefficient of variation for steady-state ibuprofen permeation from a gel formulation was reduced from 25.7% to 5.3% (n = 6). Thus, validation and refinement of the protocol combined with improved operator training can greatly enhance reproducibility in Franz cell experimentation.