A multifaceted approach is presented as a general strategy to identify new drug targets in a breast cancer stem cell-containing side population. The approach we have utilized combines side population cell sorting and stable isotope labeling by amino acids in cell culture with mass spectrometry to compare and identify proteins with differential expression profiles between side population cells, know to be enriched in cancer stem cells, and nonside population cells, which are depleted in cancer stem cells, for two breast cancer cell lines, MCF7 and MDA-MB231. Almost 900 proteins were quantified, and several important proteins in cell cycle control and differentiation were found to be upregulated in the cancer stem cell-containing side population. Most interestingly, a splice isoform of pyruvate kinase M2 as well as peroxiredoxin 6 were found to be downregulated. The differential levels of three of these proteins, thymosin beta4 (TB4), proliferation-associated protein 2G4, and SIAH-interacting protein, were validated using Western blot. Furthermore, functional validation provided clear evidence that elevated TB4 expression contributes to drug resistance in the stem cell population. Small interfering RNA silencing of TB4 led to a loss of chemoresistance in two separate breast cancer populations. These proteins likely contribute to resistance in the cancer stem cell-containing side population, and their altered expression in a tumor causes clinical resistance to chemotherapy. The ability to perform quantitative mass spectrometry has enabled the identification of a series of proteins that could serve as future therapeutic targets.