Cancer has become one of the leading causes of death, with approximately ten million people worldwide dying from cancer each year. In most cases, cancer spreads to remote organs and develops a resistance to therapy. To reduce the deadly impact of cancer, novel targets for markers for early detection are necessary. Given the notable influence of rapid chemical turnover on isotope effects, the heightened turnover rate of cholesterol in cancer offers a promising way for investigation. Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS) offers a valuable tool of tracking cholesterol dynamics. Consequently, we employed ToF-SIMS to assess cholesterol alterations, aiming to uncover potential diagnostic vulnerabilities stemming from heightened cholesterol synthesis. Our study explored the chemical profile of cholesterol influenced by cancer cell metabolism using mammary glands from mice, both with and without cancer. Results revealed a significant increase in the fractional abundance of fragment cholesterol peaks (C27H45+) in cancerous tissues, indicating dysregulated cholesterol metabolism within cancer cells. This suggests potential structural weaknesses or incomplete synthesis. Further investigation into carbon isotope incorporation suggests that the isotopic patterns might be due to the integration of heavier carbon isotopes, although these patterns could be affected by other isotopic influences. Nevertheless, understanding isotope effect of cholesterol profiles have the potential to advance our understanding of cancer biology and improve diagnostic approaches.