Tumor biomarkers assist in the early detection of cancer, act as therapeutic targets for intervention, and function as diagnostic indicators for the evaluation of therapeutic responses. To identify novel human breast cancer biomarkers, we have analyzed the protein content of lipid rafts isolated from a series of human mammary epithelial cell lines with increasing tumorigenic potential. Since lipid rafts function as platforms for protein interaction critical to several biological processes, we hypothesized that the abundance of proteins associated with proliferation, invasion and metastasis would be dysregulated in highly transformed cells. For this purpose, the MCF10A epithelial lineage, which include benign MCF10A cells, premalignant AT and TG3B cells, and malignant CA1a tumor cells, was utilized. Detergent-resistant membranes were isolated from each line and proteins were identified and relatively quantitated using iTRAQ™ reagents and tandem mass spectrometry. 57 proteins were identified, and 1667 peptide identifications, mapping to 49 proteins, contained sufficient information for semi-quantitative analysis. When comparing malignant to benign cells, we observed consistent alterations in groups of proteins, such as a 5.7-fold average decrease in G protein content (n = 5), 2.7-fold decrease in glycosylphosphatidylinositol-linked proteins (n = 7) and 3.3-fold increase in intermediate filaments (n = 9). Several of the identified proteins, including caveolin-1, filamin A, keratins 5, 6 and 17, and vimentin, are bona fide or candidate biomarkers in clinical studies, underscoring the usefulness of the MCF10A series as a model to better understand the biological mechanisms underlying cancer progression.