Glaucoma is the world's most common cause of irreversible blindness, which makes early diagnosis, with the goal of preserving vision, essential. The current medical intervention is to reduce intraocular pressure (IOP) to slow down progression of the disease. The main goal of this study was to test a novel handheld acoustic self-tonometer on humans. Methods: A sound pressure pulse generated by a loudspeaker causes the eye to vibrate. A pressure chamber is placed on the human orbit to form a coupled system comprised of the patient's eye, the enclosed air, and the loudspeaker. A displacement sensor in front of the loudspeaker membrane allows the dynamic behavior of the entire system to be detected. Results: For this clinical trial series, a prototype of the acoustic self-tonometer principle was applied. The resulting membrane oscillation data showed sensitivity of patient IOP, but direct allocation of the measured damping and frequency to the IOP was not significant. For this reason, an artificial neural network was used to find relationships among the subjects' biometric eye parameters in combination with the self-tonometer data for the IOP reference. An expanded measurement uncertainty (k p = 2) equal to 6.53 mm Hg was determined for the self-tonometer in a Bland-Altman analysis using Goldmann applanation tonometer reference measurements. Conclusions: The usability and success rate of producing valid measurement values with the device during self-measurements by test subjects was nearly 92%. The crosssensitivities observed require compensation in a possible redesign phase to reduce the measurement uncertainty by at least 25% to the maximum of 5 mm Hg required to seek medical device approval. Translational Relevance: Building on successful laboratory experiments with pig eyes, this article reports the results of testing the acoustic tonometer on humans.