In vitro release tests have been widely used to assess performance of topical products. Any interaction should not be observed between drug and synthetic membrane, but there is no conclusive information about relationship of membrane type and properties of drug substances. In this study, four types of membranes (regenerated cellulose, cellulose acetate, mixed cellulose ester, silicone) were used to evaluate in vitro release of hydrophilic, moderately lipophilic and highly lipophilic drug substances from their topical preparations. The relationship between membrane type and drug characteristics was investigated using Strat-M™ membrane resembling to human skin, and excised porcine skin for in vitro permeation studies. The release data revealed that acceptor/donor groups in drug substance and synthetic membrane could hinder drug release through membrane. Mixed cellulose ester or cellulose acetate membranes should be opted for in vitro release studies of drugs consisting of abundant, strong acceptor/donor groups. In vitro permeation data showed that drug lipophilicity might come into prominence for these studies. Strat-M™ membrane could be more useful to estimate permeability of lipophilic molecules rather than hydrophilic ones. The results suggest that molecular structure and lipophilicity of drug could be considered to select synthetic membrane used for in vitro release and permeation studies.