This study aimed to evaluate the static and dynamic pupil changes, and light sensitivity following a single dose of low-dose atropine at concentrations of 0.01%, 0.025%, and 0.05% over a 24 h period. Healthy young adults (20–22 years; n = 25) participated in this randomized, double-blind study. Each participant received one of three atropine concentrations in a masked fashion. Baseline mesopic and dynamic pupil sizes were measured at various post-instillation intervals (5 min, 30 min, 1 h, 2 h, 4 h, and 24 h). A minimum 48 h washout period was observed between treatments. Subjective light sensitivity was assessed using the Visual Light Sensitivity Questionnaire (VLSQ-8) at 24 h. All atropine concentrations caused significant pupil dilation (p < 0.001), with the 0.05% concentration producing the greatest dilation (peak mesopic size: 7.4 mm, p < 0.001) and the slowest recovery at 24 h (6.4 mm, p < 0.001). The dynamic pupil constriction range was most restricted with 0.05% (1.7 mm, p < 0.05), compared to 0.025% (2.2 mm) and 0.01% (2.6 mm). Subjective symptoms, including light sensitivity and glare, followed a dose-dependent pattern (p < 0.05). In 60% participants, 0.05% caused the most symptoms, while in 70% participants, 0.01% caused the least. Despite significant pupil dilation, the pupil center coordinates did not shift significantly along the horizontal or vertical axes (p > 0.05). Low-dose atropine induced dose-dependent pupil dilation and light sensitivity; 0.05% atropine caused the most pronounced effects. These findings underscore the importance of tailoring the atropine dosage to balance its efficacy and tolerability. Further studies are needed to explore the long-term impact of repeated dosing on pupillary behavior and subjective symptoms.