Background: Progressive supranuclear palsy (PSP) is a neurodegenerative disorder that, especially in the early stages of the disease, is clinically difficult to distinguish from Parkinson's disease (PD). Objective: This study aimed at assessing the use of eye-tracking in head-mounted displays (HMDs) for differentiating PSP and PD. Methods: Saccadic eye movements of 13 patients with PSP, 15 patients with PD, and a group of 16 healthy controls (HCs) were measured. To improve applicability in an inpatient setting and standardize the diagnosis, all the tests were conducted in a HMD. In addition, patients underwent atlas-based volumetric analysis of various brain regions based on high-resolution MRI. Results: Patients with PSP displayed unique abnormalities in vertical saccade velocity and saccade gain, while horizontal saccades were less affected. A novel diagnostic index was derived, multiplying the ratios of vertical to horizontal gain and velocity, allowing segregation of PSP from PD with high sensitivity (10/13, 77%) and specificity (14/15, 93%). As expected, patients with PSP as compared with patients with PD showed regional atrophy in midbrain volume, the midbrain plane, and the midbrain tegmentum plane. In addition, we found for the first time that oculomotor measures (vertical gain, velocity, and the diagnostic index) were correlated significantly to midbrain volume in the PSP group. Conclusions: Assessing eye movements in a HMD provides an easy to apply and highly standardized tool to differentiate PSP of patients from PD and HCs, which will aid in the diagnosis of PSP. Copyright © 2021 Herwig, Agic, Huppertz, Klingebiel, Zuhorn, Schneider, Schabitz and Rogalewski.