Folic acid is an essential B vitamin, the metabolism of folic acid via one carbon metabolism results in the production of important components for the cell, such as DNA bases and methyl donor groups. The importance of mitochondrial one carbon metabolism has recently been highlighted with the discovery of the novel enzyme Dihydrofolate Reductase like 1 (DHFRL1) and the association of other mitochondrial enzymes, in particular Methylenetetrahydrofolate dehydrogenase (NADP+ dependent) 1-like (MTHFD1L) with rapid proliferation and mortality in cancer. The novel DHFRL1 enzyme has been shown to be a much less active enzyme with a reduced affinity for dihydrofolate relative to DHFR. The MTHFD1L enzyme is responsible for the last step in the production of formate for cytoplasmic one carbon metabolism. Due to MTHFD1L’s associations with rapid proliferation rate in cancer and mortality, a biomarker for its expression is desirable. Formate analysis by Gas chromatography–mass spectrometry (GC-MS) in Human Embryonic Kidney (HEK) 293 cells with modulated MTHFD1L expression demonstrated that knocking down the gene resulted in reduced formate levels and reduced cell growth. Similarly overexpressing the MTHFD1L gene in HEK 293 cells resulted in an increased formate level and growth rate relative to the controls. Investigation was undertaken into the amino acid differences between DHFR and DHFRL1 to begin to understand their functional relevance. It was identified that the arginine at amino acid position 24 may result in DHFRL1 having an altered structure, which may account in part for DHFRL1’s reduced affinity for dihydrofolate. In addition, DHFRL1 and other mitochondrial folate enzymes, MTHFD1L, Methylenetetrahydrofolate dehydrogenase (NADP+ dependent) 2 (MTHFD2), Serine Hydroxymethyltransferase (mitochondrial isoform) 2 (SHMT2) were found to be up-regulated in a metastatic cancer cell line but their cytosolic paralogues showed no such up-regulation. The results presented provide further evidence of the mitochondrial driven role in cancer progression and are supportive for the use of formate as a biomarker for mitochondrial gene up-regulation.