Purpose: To test whether adding topical phenylephrine 5% to tropicamide 0.5% eye drops in the protocol for pupil dilation affects the retinal vessel oximeter measurements in patients with glaucoma. To test whether phenylephrine 5% has an influence as a vasoconstrictor on the retinal vessel width and can improve the proportion of high-quality retinal images in patients with glaucoma. Methods: Retinal images of 66 patients with chronic open-angle glaucoma were obtained before and after the administration of phenylephrine 5% eye drops to patients already dilated with tropicamide 0.5% with the Oxymap Retinal Oximeter (Oxymap ehf, Reykjavik, Iceland). Specialized software, Oxymap Analyzer, analysed the images and measured the oxygen saturation and vessel diameter. Oxygen saturation was measured in first- and second-degree vessels. A Mann-Whitney U-test was used to compare both groups. Quality of the images was assessed, and a Fisher's exact test was used to compare the proportion of high- and poor-quality images. Results: There was no significant difference in arterial and venous oxygen saturation in patients with glaucoma whether dilated by tropicamide alone or a combination of tropicamide and phenylephrine (97 ± 6% versus 96 ± 5%, p = 0.88 for arterial saturation and 66 ± 6% versus 67 ± 6%, p = 0.78 for venous saturation, n = 27). There was no significant difference in vessel diameter between both conditions for the different vessels (p = 0.61 for arterial saturation and p = 0.51 for venous saturation, n = 27). The proportion of high-quality images was significantly higher after the combination regimen compared with tropicamide only (p = 0.0001). Conclusion: The addition of topical phenylephrine 5% after tropicamide 0.5% improved the proportion of high-quality retinal oximetry images without influencing the retinal oxygen saturation values or the retinal vessel diameter in patients with glaucoma.