Updated project metadata. Colorectal cancer (CRC) is projected to become the third most diagnosed and third most fatal cancer in the United States by 2024, with early onset CRC on the rise. Research is constantly underway to discover novel therapeutics for the treatment of various cancers to improve patient outcomes and survival rates. Recently, fatty acid synthase (FAS) has become a druggable target of interest for the treatment of many different cancers. FAS inhibitors have been developed over the years with mixed success. One such inhibitor, TVB-2640, has gained popularity for its high specificity for FAS and has even entered a phase 1 clinical trial for the treatment of solid tumors. However, the distinct molecular differences that occur upon inhibiting FAS have yet to be understood. Here, we conduct time course proteomics and phosphoproteomics on analyses HCT 116 and HT-29 CRC spheroids inhibited with either a generation 1 (cerulenin) or generation 2 (TVB-2640) FAS inhibitor. Proteins involved in lipid metabolism and cellular respiration were altered in abundance. It was also observed that proteins and enzymes involved in ferroptosis—an iron mediated form of cell death—were altered. These results show HT-29 spheroids exposed to cerulenin or TVB-2640 are undergoing a ferroptotic death mechanism.