Adequate levels of folates are essential for homeostasis of the organism, prevention of congenital malformations, and the salvage of predisposed disease states. They depend on genetic predisposition, and therefore, a pharmacogenetic approach to individualized supplementation or therapeutic intervention is necessary for an optimal outcome. The role of folates in vital cell processes was investigated by translational pharmacogenetics employing lymphoblastoid cell lines (LCLs). Depriving cells of folates led to reversible S-phase arrest. Since 5,10-methylenetetrahydrofolate reductase (MTHFR) is the key enzyme in the biosynthesis of an active folate form, we evaluated the relevance of polymorphisms in the MTHFR gene on intracellular levels of bioactive metabolite, the 5-methyltetrahydrofolate (5-Me-THF). LCLs (n = 35) were divided into low- and normal-MTHFR activity groups based on their genotype. They were cultured in the presence of folic acid (FA) or 5-Me-THF. Based on the cells’ metabolic activity and intracellular 5-Me-THF levels, we conclude supplementation of FA is sufficient to maintain adequate folate level in the normal MTHFR activity group, while low MTHFR activity cells require 5-Me-THF to overcome the metabolic defects caused by polymorphisms in their MTHFR genes. This finding was supported by the determination of intracellular levels of 5-Me-THF in cell lysates by LC-MS/MS. FA supplementation resulted in a 2.5-fold increase in 5-Me-THF in cells with normal MTHFR activity, but there was no increase after FA supplementation in low MTHFR activity cells. However, when LCLs were exposed to 5-Me-THF, a 10-fold increase in intracellular levels of this metabolite was determined. These findings indicate that patients undergoing folate supplementation to counteract anti-folate therapies, or patients with increased folate demand, would benefit from pharmacogenetics-based therapy choices.