PRéCIS:: Small but significant decreases in OCTA-measured circumpapillary capillary density were observed in healthy eyes dilated with 2.5% phenylephrine/0.5% tropicamide. Though likely clinically insignificant, ophthalmologists should consider these changes when interpreting OCTA results from dilated eyes.; Purpose: To investigate the effect of pupil dilation using 2.5% phenylephrine and 0.5% tropicamide on quantitative assessment of retinal microvasculature using OCTA.; Methods: OptoVue AngioVue high density (HD) and non-HD OCTA macula and optic nerve head (ONH) images were obtained at 15-minute intervals pre- and post-dilation in 26 healthy participants (mean age: 40.0; 95% CI=33.9, 46.1▒y). Superficial macular vessel density (VD) was measured in the whole image (wiVD) and the parafoveal region (pfVD). Optic nerve head capillary density was measured in the whole image (wiCD) and the circumpapillary region (cpCD). Differences between pre- and post-dilation densities were assessed using linear mixed effects models to account for within-patient correlation.; Results: Instillation of dilating drops resulted in a small but statistically significant reduction in non-HD ONH wiCD of 0.6%, from a mean of 45.2% (95% CI=41.9%, 48.4%) to 44.6% (41.4%, 47.8%) (P=0.046). A similar reduction in non-HD ONH cpCD of 0.8% also was observed, from a mean of 49.3% (45.3%, 53.3%) to 48.5% (44.5%, 52.4%) (P=0.025). No post-dilation decreases in macular vessel density or HD ONH capillary density were observed.; Conclusion: Pupil dilation using topical 2.5% phenylephrine and 0.5% tropicamide results in a small but statistically significant reduction in non-HD ONH whole image and circumpapillary capillary densities in healthy eyes. The observed reduction likely is not clinically significant because the observed reduction was within the previously reported range of measurement variability. Further studies should consider investigating these effects in non-healthy eyes with glaucoma and media opacities, as well as older individuals.