Phenolic are secondary plant metabolites and bioactive compounds, which may act as antioxidants due to their free radical scavenging properties. The productions of plant phenolic are of great importance for pharmaceutical, food and nutraceutical industries. They are used as food colorants; nutraceuticals and generally have the capacity to improve overall human health. Grape is a natural source of polyphenols, which possess remarkable nutraceuticals and phytochemical properties. The overall goal of this research was to conduct a feasibility study for the production of dietary antioxidants by in vitro cell culture of the American native grape, Vitis aestivalis (Michx.) Cynthiana cv. The outcome will permit a molecular farming of the cell line that produces the highest level of dietary antioxidants and to establish a novel sustainable resource for the food, pharmaceutical and nutraceuticals industries. The in vitro cell lines of Vitis aestivalis Michx. Cynthiana cv. was initiated from the early veraison stage of grape berries using a laboratory protocol. The initial callusogenesis appeared after 4 weeks. The visual color difference in callus morphology was used for selection of various in vitro lines. Four assays: 2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical scavenging; ferrous reducing antioxidant power (FRAP); cupric reducing antioxidant capacity (CUPRAC); and trolox equivalent antioxidant capacity (TEAC) were used to determine the antioxidant capacity, phytochemical analysis of investigated cell lines, and growth dynamics of initiated cell suspensions were determined. Biochemical analysis confirmed variation of the production of secondary metabolites in the callus cell lines, which permitted the selection of line 15, 11, 1, 16 and 6 containing, indicating the highest level of antioxidant capacity, phenolic, monomeric anthocyanin, flavonols, and proanthocyanin content respectively. Also, growth dynamics was determined line 3, 7, 12, and 17 base on their performance when suspended in B-5 medium.