Objective This study compared two methods for preparing retinal pigment epithelium (RPE) flat mounts and identified the difficulties of each method. The influences of each method on the RPE cell morphology and the integrity of RPE flat-mount were also presented. Methods In this experimental study, in the first method, direct flat mounts were prepared from rat eyes that were removed and fixed for 30 min in phosphate-buffered saline containing buffered 4% paraformaldehyde (PFA). The anterior segment of each eye and retina were removed. The remaining eye cup containing the RPE was radially cut into 4 to 6 pieces from the periphery to the optic nerve head. In the second method in which the RPE was isolated, the eye was removed and fixed as described above except that fixation continued overnight. For the isolated flat-mount technique, the anterior segments and retina were removed. The RPE-Bruch membrane complex was then carefully isolated from the remaining eye cup, and the upward facing RPE flat mount was formed by several relaxing radial cuts. Results The isolated flat-mount method could be used to obtain the whole RPE flat-mount, and the morphology of the RPE cells could be maintained. However, the preparation process of the direct flat-mount method could cause loss of the RPE cells, especially those at the edge of the sclera. Conclusion The direct RPE flat-mount method is simple and easy to perform except that the RPE cells at the edge of sclera are disrupted. The isolated RPE flat-mount method is relatively more difficult, but RPE cells are well preserved.