Tight junctions are critical determinants of epidermal barrier integrity, with claudin proteins serving as key structural and functional regulators of paracellular permeability in human keratinocytes. Alterations in tight junction organization and claudin expression are associated with inflammatory skin diseases, defective wound healing, and barrier dysfunction; however, standardized experimental approaches for their reliable assessment in epidermal keratinocyte cultures remain limited. This chapter describes an optimized and reproducible experimental workflow for the evaluation of tight junction integrity and claudin expression in cultured human epidermal keratinocytes. The method combines transepithelial electrical resistance (TEER) measurements for functional barrier assessment, fluorescent tracer–based paracellular permeability assays, and immunofluorescence microscopy for spatial localization of tight junction proteins, including claudins, occludin, and ZO-1. Quantitative analysis of claudin expression is further achieved using Western blotting and real-time PCR. Key experimental variables such as calcium switch conditions, seeding density, keratinocyte differentiation status, and antibody validation are highlighted to ensure robustness and reproducibility. This integrated protocol provides a reliable platform for investigating epidermal tight junction dynamics in skin barrier research, disease modeling, toxicological testing, and therapeutic evaluation.