The up-regulation of endogenous fatty acid and complex lipid synthesis is a characteristic feature of breast cancer, but their role in carcinogenesis remains largely unknown. Phospholipids mediate important cellular processes such as membrane organization, intercellular communication, cell proliferation, and apoptosis. So, understanding mechanisms of tumor growth requires elucidating the lipid composition of cancer cells as a function of gene expression. In addition, genetic classification of breast cancer cells is important for diagnostics and for the development of personalized treatment regimens. At present, lipidomics of cancer cells is typically carried out using high performance liquid chromatography-mass spectrometry after application of various solvent extraction methods. These approaches involve relatively time consuming separation steps. Herein, we describe a desorption electrospray ionization mass spectrometry (DESI-MS) cell-based workflow that allows direct characterization and classification of breast cancer cell lines having different genetic mutations based on their lipid compositions. In this approach, cultured cells were deposited on microscope slides to form a small pellet of a few millimeters in diameter, which was then analyzed using DESI-MS coupled to an LTQ-Orbitrap XL mass spectrometer. Using this approach, we characterized the lipid compositions of various breast cancer cell lines and showed that there is a general increase in phosphatidylinositols when compared to an immortalized normal cell line. These results agree with previous studies showing that PI3K/Akt pathways are up-regulated in breast cancer, indicating that cell-based DESI-MS assays are powerful tools for probing intrinsic disease mechanisms in vitro.