Purpose. We examined in vitro the potential evaporationretarding effect of the tear film lipid layer (TFLL). The artificial TFLL compositions used here were based on the present knowledge of TFLL composition. Methods. A custom-built system was developed to measure evaporation rates at 35°C. Lipids were applied to an air-water interface, and the evaporation rate through the lipid layer was defined as water loss from the interface. A thick layer of olive oil and a monolayer of long-chain alcohol were used as controls. The artificial TFLLs were composed of 1 to 4 lipid species: polar phosphatidylcholine (PC), nonpolar cholesteryl ester, triglycerides, and wax ester (WE). Brewster angle microscopy (BAM) and interfacial shear rheometry (ISR) were used to assess the lateral structure and shear stress response of the lipid layers, respectively. Results. Olive oil and long-chain alcohol decreased evaporation by 54% and 45%, respectively. The PC monolayer and the fourcomponent mixtures did not retard evaporation. WE was the most important evaporation-retardant TFLL lipid (~20% decrease). In PC/WE mixtures, an ~90% proportion of WE was required for evaporation retardation. Based on BAM and ISR, WE resulted in more condensed layers than the nonretardant layers. Conclusions. Highly condensed, solid-like lipid layers, such as those containing high proportions of WEs, are evaporationretardant. In multi-component lipid layers, the evaporationretardant interactions between carbon chains decrease and, therefore, these lipid layers do not retard evaporation.