Giant unilamellar vesicles (GUVs of diameter 5-25 μm) of soy phosphatidylcholine (PC), resistant to intense light exposure (400-440 nm, ~15 mW·mm(-2)), underwent budding when containing chlorophyll a (Chla) in the lipid bilayer ([PC]:[Chla] = 1500:1). On the basis of image heterogeneity analysis using inverted microscopy, a dimensionless entropy parameter for the budding process was shown to increase linearly during an initial budding process. Lipophilic β-carotene (β-Car, [PC]:[β-Car] = 500:1) reduced the initial budding rate by a factor of 2.4, while the hydrophilic glycoside rutin ([PC]:[rutin] = 500:1) had no effect. Chla photosensitized oxidation of PC to form linoleoyl hydroperoxides, further leading to domains of higher polarity in the vesicles, is suggested to trigger budding. The average dipole moment (μ) of linoleic acid hydroperoxides was calculated using density functional theory (DFT) to have the value of 2.84 D, while unoxidized linoleic acid has μ = 1.86 D. β-Carotene as a lipophilic antioxidant and singlet-oxygen quencher seems to hamper oxidation in the lipid bilayers and delay budding in contrast to rutin located in the aqueous phase. The effect on budding of GUVs as a detrimental process for membranes is suggested for use in assays for evaluation of potential protectors of cellular integrity and functions under oxidative stress.