Protein phosphorylation is a reversible protein post-translational modification which likes a molecular switch controlling important biological processes such as cell division, growth, differentiation, and death. Its misregulation is often associated with many human diseases, including cancer12. The research results from Choi et al. showed that sesquiterpene lactones can act as phosphatase inhibitors13. Therefore, we speculated that the cytotoxicity of VOSL or CD towards human breast cancer cells should be associated with protein phosphorylation pathways. Developments of phosphopeptide enrichment technologies along with improvements in mass spectrometer sensitivity, protein database and bioinformatics algorithms, have facilitated the qualitative and quantitative analyses of phosphopeptides from complex cell extracts and greatly revolutionized the fields of cell biology and cell signaling14. Currently, TiO2 has been considered as the most effective enrichment material for phosphopeptides15, and isobaric tags for relative and absolute quantification (iTRAQ) technology has been widely used to proteome research. In this study, we explored the anti-breast cancer molecular mechanism of VOSL and CD through TiO2-based enrichment of phosphopeptides and iTRAQ-based liquid chromatography and tandem mass spectrometry (LC-MS/MS) proteomics, coupled with Western blot validation.