Polarized epithelial cells are compartmentalized into apical and basement membranes with asymmetrically distributed proteins. This study aimed to establish a method for culturing epithelial cells at the fluorinated oil (Novec-7500) microdroplet surface for the formation of epithelial polarity, which is desirable for regenerative medicine and drug discovery research. Microdroplet surfaces treated with fibronectin, which regulates a variety of cell behaviors through direct interactions with cell surface integrin receptors, were prepared for culturing epithelial cells. The cells adhered rapidly to the fibronectin-coated fluorinated oil-medium interface and reached confluence. However, as the culture time progressed, the cells began to detach from the microdroplet surface. To promote adhesion to the microdroplet interface, the cells were exposed to the Rho-associated protein kinase inhibitor Y27632, which increased the frequency of microdroplets with cells adhering to the liquid-liquid interface by 1.63-fold. However, continuous exposure to Y27632 caused the cells to detach from the microdroplet surface. When the drug was switched from Y27632 to forskolin, which enhances cell-cell and cell-substrate adhesion, the cells remained as a monolayer on the microdroplet interface. Na