Drug absorption after oral administration mostly occurs in the intestine, and is controlled mainly by the aqueous solubility and the intestinal permeability of the drug. In modern drug discovery, the proportion of lipophilic drug candidates with poor water solubility has increased, and these lipophilic drugs are known to undergo membrane retention during transport across the cell membrane, depending on the hydrophobic interaction between the drug and lipid molecules. Hence, a precise and effective permeability assay for lipophilic drug compounds, which can also quantify membrane retention, is required. In this study, we developed a permeability assay for lipophilic drugs with poor water solubility using a freestanding lipid bilayer. The lipid bilayer was created within a UV cuvette, and the number of transported molecules through the bilayer was estimated by measuring the UV absorbance over time. We then measured the permeability of six tested compounds, and there was a significant difference in permeability between the Biopharmaceutics Classification System (BCS) class 2 and class 4 compounds. In addition, the fraction of molecules trapped in the lipid bilayer were estimated for each compound as well. As a result, the drugs with higher lipophilicity were found to undergo more membrane retention, which agrees with the previous reports, supporting that our model lipid membrane system could also be applied to investigate the drug-lipid interaction.