Ocular tissues function as biological barriers that hinder drug delivery, depending on the target tissue and route of administration, and must be overcome to achieve the desired therapeutic effect. Penetration enhancers have long been investigated to improve corneal drug penetration via eye drop instillation; however, further development is warranted owing to potential safety concerns. In the present study, we focused on cell-penetrating peptides (CPPs) as a penetration enhancer to address the requirements and explored CPP candidates suitable for corneal drug delivery. Using a reconstructed human corneal epithelial tissue model, LabCyte CORNEA-MODEL24 as an alternative to animal testing that is expected to have higher reproducibility than extracted eyeballs and octa-arginine (R8) as a representative model CPP with simple structure, we investigated the enhancement of 6-carboxyfluorescein (6-FAM) uptake by fluorescence imaging and the potential of eye irritation by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Also, surface plasmon resonance (SPR) evaluated the interaction between R8 and model compounds, suggesting that the stronger interaction could facilitate the corneal uptake of compounds. A comparative screening study of corneal uptake using various CPPs showed that the CPPs other than R8 also have the potential to enhance the corneal uptake of 6-FAM. In particular, penetratin (PNT) showed stronger fluorescence intensity. Through these findings, this manuscript provides beneficial information for the development of a novel corneal penetration enhancer with CPPs. In the future, it is expected that the basic findings with R8 will be verified to be applicable to other CPPs for development as penetration enhancers for eye drop formulation.