As a result of polymer materials development and the use of additive manufacturing technologies, gear wheels made of polymer materials are becoming widespread in many areas of the industry. In recent years, determining the dynamic behavior of polymer gears has gained significant importance because it is desired to carry more loads and operate at higher speeds. Since it is one of the most critical factors affecting dynamic behavior, tooth stiffness should also be determined. In this study, the single-tooth stiffness (STS) of polymer gears with symmetrical and asymmetrical profile was measured experimentally with a unique test setup. Force was applied to three different points on the tooth, and the resulting deflection was measured with the help of linear variable differential transformer and a high-speed camera. Using the obtained deflection values, STS of the polymer tooth was calculated depending on the pressure angle. The experimental results are also compared with the finite element model created, and it is found that the results are matched well. As a result of the study, it is determined that the drive-side pressure angle of the polymer gear increased from 20 degrees to 32 degrees, and the tooth stiffness increased by approximately 10.8%.