Introduction: The pupillary light reflex (PLR) is a well-validated biomarker for neurologic monitoring and a decision-making tool for traumatic brain injury patients. We studied a machine learning-based mobile pupillometry platform in patients with acute ischemic stroke (AIS) with a large-vessel occlusion (LVO) prior to thrombectomy, compared to healthy volunteers. Methods: Pupillometry measurements were conducted with a mobile pupillometry platform (PupilScreen) and a digital infrared pupillometer (NeurOptics) for both pre-thrombectomy AIS subjects and healthy volunteers at an academic medical center. To correct for subject age differences, comparisons used the absolute inter-eye difference in each parameter for each subject by measuring the right:left (R:L) eye ratio absolute distance away from 1. Inter-eye difference means across subjects between AIS and healthy cohorts were analyzed for PLR changes in the presence of acute LVO using a t-test for independent means. Results: Seven AIS patients (4 female, 3 male; 1 Hispanic, 6 Caucasian; mean age 60.9 years) and 32 healthy patients (19 female, 13 male; 1 Hispanic, 2 African American, 3 Native American, 5 Asian, 21 Caucasian; mean age 34.4) were enrolled. All LVOs were of the middle cerebral artery, with 3 on the left and 4 on the right. NPi value was above 3 (briskly reactive) for all subjects. However, the smartphone pupillometer demonstrated a statistically significant (p<0.05) increase in mean inter-eye differences for maximum diameter (mean inter-eye difference of 0.14 vs 0.03), minimum diameter (0.21 vs 0.03), and percent change (0.23 vs 0.05). The mean inter-eye differences were lateralized to the side of the LVO for maximum and minimum diameters (right LVO, max pre vs post R:L 0.95 vs 1.0, min 0.91 vs 0.98) (left LVO, max 0.98 vs 0.96, min 1.15 vs 0.93), and were increased in latency and mean constriction velocity whereas they were decreased in maximum constriction velocity and mean dilation velocity, but this was not statistically significant in this cohort. Conclusions: Patients with AIS from LVO demonstrate inter-eye differences in PLR parameters, and mobile pupillometry could be used as a pre-intervention biomarker of AIS in future study. Further investigation in a larger cohort is necessary.