Purpose:To examine riboflavin concentrations in corneas and aqueous humor from rabbits with standard and transepithelial methods and iontophoresis without disrupting the integrity of the corneal epithelium before corneal collagen cross-linking.Methods:Twenty-four eyes of 12 adult New Zealand rabbits were used. They were assigned to 4 groups, each including 6 eyes. Group 1 was exposed to the standard method and given riboflavin 0.1% after epithelial debridement. Group 2 was exposed to the transepithelial method and given benzalkonium chloride (BAC), ethylenediaminetetraacetic acid (EDTA), trometamol (TRIS), hydroxypropylmethylcellulose (HPMC), and riboflavin 0.2% 3 times at 1.5-minute intervals followed by riboflavin 0.2%. Group 3 was given riboflavin 0.1% by using 1-mA electric current for 10 minutes with the help of iontophoresis without using substances disrupting the integrity of the corneal epithelium. Group 4 received the same treatment as did group 3, except that it was given riboflavin 0.2%. Following these treatments, riboflavin concentrations in aqueous humor and corneas were measured with ultraperformance liquid chromatography coupled with electrospray ionization tandem mass spectrometry (UPLC-ESI-MS/MS).Results:Riboflavin concentrations in the cornea and aqueous humor were higher in group 1 (42.4 5.4 g/g) than in the other groups. They were significantly higher in group 4 (34.2 +/- 6.6 g/g) than in group 2 (24.4 +/- 1.2 g/g) (P = 0.009) and group 3 (23.6 +/- 6.1 g/g) (P = 0.026). There was not a significant difference in corneal riboflavin concentrations between group 2 and group 3 (P = 0.937).Conclusions:Intrastromal and aqueous riboflavin concentrations after administration of riboflavin 0.2% through iontophoresis without disrupting the integrity of the corneal epithelium were lower than those after the standard method, but higher than those after the transepithelial method. In this study, in which riboflavin concentrations were measured with a very sensitive method, iontophoresis was observed to increase the transmission of riboflavin molecules into the cornea without using substances disrupting epithelial integrity.