The effects of glycation on skin permeation and accumulation of compounds were evaluated using an in vitro glycated skin model. Glycation of the skin of hairless mice was induced using vertical diffusion cells and incubation with phosphate-buffered saline containing 50 mM glyoxal for 24 h. Flux and accumulation in the skin were determined by applying hydrophilic and lipophilic molecules (Sodium fluorescein; FL-Na and Nile red, respectively) to this in vitro glycated skin model. Furthermore, to investigate the effect of glycation on epidermal–dermal barrier properties, we conducted diffusion experiments with FL-Na and fluorescein isothiocyanate-dextran using stratum corneum (SC)-stripped glycated skin. The in vitro glycated skin model demonstrated characteristic glycation alterations like a yellowish change in skin color and surface roughness. For low-molecular weight (MW) hydrophilic molecules, flux across glycated full-thickness skin was higher than that across normal skin, although there was no difference with lipophilic molecules. However, glycated epidermis–dermis showed lower flux, and the difference increased with the MW of the compound. Furthermore, the amount of high-MW hydrophilic molecules accumulated in glycated epidermis–dermis was decreased. These results suggest that glycated SC and epidermis–dermis differentially regulate the permeability of hydrophilic molecules and highlight the importance of controlling drug delivery by modifying the formulation or method of application depending on skin condition.