Estrogen-triggered nuclear transcription factor, estrogen receptor (ER) is expressed in about 70% of breast cancer. ER mediates various cellular signaling events including the genomic regulation of target genes that affect reproduction, development, and general metabolism. The antiestrogen tamoxifen is currently administered to patients in order to induce regression of the tumor growth. However, upon continued administration, breast cancer patients develop resistance to tamoxifen. In addition, calcium binding proteins such as, calmodulin (CaM) which are significantly overexpressed in cancer cells, can activate transcription of target genes by directly binding to ER in lieu of estrogen. Calmodulin antagonists (w7 and melatonin) have been shown to significantly inhibit human estrogen receptor alpha (hERalpha) transcriptional activity. In search of a novel anti-cancer agent, we generated a library of interaction profiles of human ER-EF hand protein complexes and identified S100B and S100P (high sequence and structure homology with CaM) as potential targets. Furthermore, S100P is shown to mediate tamoxifen-acquired resistance and cell migration capacity in MCF-7 breast cancer cells. Our objective is to develop peptidomimetics and small molecules to block ER-S100 interactions using an integrated proteomics-assisted drug discovery approach. Briefly, using the fold information and contact regions obtained from the FT-ICR mass spectrometry, 3D structures of ER-S100 protein complexes were reconstructed. These structures of ER best represent the conformational state sensed by the specific interacting partner. ER-S100 complex is distinct from CaM. The lack of the connective peptidic region between the EF hand pairs in S100 attributed to less interaction coverage than CaM. The identified ER conformation-sensing regions of the interacting EF hand proteins were used to develop a library of top peptidomimetics and potential small molecule sensors targeting the interfaces of ER-S100 interactions. We have evaluated their ability to block specific ER-S100 interactions in a high throughput fluorescent polarization based screen, to inhibit cancer cell proliferation and ER-mediated transcription of target genes. We believe that the ensemble of ER-EF hand protein complexes generated by our integrated proteomics-assisted protein interaction profiling will shed light on the lingering issue of hormone independent activation of ER at the molecular level and novel therapeutics targeting ER-S100 interactions.