Activated choline phospholipid metabolism is a hallmark of cancer. Aggressive breast cancers are characterized by high tumoral phosphocholine and glycerophosphocholine. In our ongoing efforts of evaluating the glycerophosphodiesterases GDPD5 and GDPD6 as cancer treatment targets, we have systemically injected mice growing orthotopic triple-negative MDA-MB-231 breast tumors with lentiviral vectors that silence the GDPD5 or GDPD6 genes as compared to mice injected with control viruses. We have analyzed extracted tumor tissue by means of high-resolution 1H MRS-based metabolomics. Differences in tumor growth and metabolic profiles were observed following silencing of GDPD5 and GDPD6 genes when compared to control mice.