Retinal pigment epithelium, which forms the outer blood-retinal barrier, is a critical barrier for transport of drugs to the retina. The purpose of this study was to develop a pigmented MDCK (P-MDCK) cell line as a rapidly established in vitro model for the outer blood-retinal barrier to assess the influence of melanin pigment on solute permeability. A melanin synthesizing P-MDCK cell line was developed by lentiviral transduction of human tyrosinase and p-protein genes in MDCK (NBL-2) cells. Melanin content, tyrosinase activity (conversion of L-dopa to dopachrome), and transepithelial electrical resistance (TEER) were measured. Expression of tyrosinase protein and p-protein in P-MDCK cells was confirmed by confocal microscopy. Effect of l-tyrosine (0 to 2 mM) in culture medium on melanin synthesis in P-MDCK cells was evaluated. Cell uptake and transepithelial transport of pigment-binding chloroquine (Log D = 1.59) and a negative control salicylic acid (Log D = -1.14) were investigated. P-MDCK cells expressed tyrosinase and p-protein. Tyrosinase activity was 4.5-fold higher in P-MDCK cells compared to wild type MDCK cells. The transepithelial electrical resistance stabilized by day 4 in both cell types, with the TEER being 958 ± 33 and 964 ± 58 Ω·cm(2) for P-MDCK and wild type cells, respectively. Melanin content in P-MDCK cells depended on the concentration of l-tyrosine in culture medium, and increased from 3 to 54 μg/mg protein with an increase in l-tyrosine content from 0 to 2 mM. When the cells were grown in 2 mM l-tyrosine, uptake of chloroquine was 2.3-fold higher and the transepithelial transport was 2.2-fold lower in P-MDCK cells when compared to wild type MDCK cells. No significant difference was observed for both cell uptake and transport of salicylic acid. We developed a P-MDCK cell line with tunable melanin synthesis as a rapidly developing surrogate for retinal pigment epithelium.