Breast cancer is the most common cancer in women worldwide despite recent progress in breast cancer diagnosis and therapy. Thus, it remains crucial to explore novel approaches for its management. Sphingolipids have emerged as important regulators of cancer cell fate: ceramide has been shown to be pro-apoptotic, while sphingosine-1 phosphate (S1P) induces cell growth and survival. This research explores the roles of sphingolipid metabolites by evaluating five Drosophila melanogaster mutants with altered endogenous levels of ceramide, S1P, sphingosine and sphingadiene. Isolating, purifying and measuring lipid extracts from Drosophila allows the assessment of how different levels of sphingolipids can affect the growth of breast cancer cells by measuring their cytotoxicity, ability to generate ROS, and their effect on cell proliferation. The results indicate that the Sply, SK-1 and SK-2 lipid extracts were cytotoxic to breast cancer cells and stimulated ROS production in vitro. These results confirm the importance of investigating sphingolipid metabolites for chemotherapeutic development.