Abstract Triple-negative breast cancer (TNBC) is a type of breast cancer that does not express the estrogen receptors (ER), progesterone receptors (PR), and human epidermal growth factor receptor 2 (HER2) protein which accounts for 15–20 % of all breast cancer cases. TNBC tumors are generally larger, are of higher grade, and are more aggressive than other breast cancer types. TNBC is unlikely to respond to hormonal therapy medicines, including tamoxifen and aromatase inhibitors. TNBC also is unlikely to respond to medicines that target the HER2 protein, such as Herceptin (trastuzumab). Treatment of TNBC patients has been challenging due to the lack of molecular targets. Therefore, there is a critical unmet need to develop more effective therapies for TNBC and drug-resistant breast cancers. The development of new treatments would require radically different approaches that rely on enzymatic reactions specific to TNBC cells rather than the cell receptors. This proposal hypothesizes that properly designed peptide substrates of Eyes Absent enzyme (EYA) can selectively inhibit TNBC cell growth by a self-assembling process. The central hypothesis is that tyrosine phosphatase activity of EYA in TNBC could specifically convert the peptide therapeutics into nanofibers and induce controlled apoptosis of TNBC cells. The preliminary data suggested that a modified sequence of peptides with only one added amino acid could be de-phosphorylated by EYA and self-assemble into nanostructures to inhibit the growth of TNBC cells (MDA-MB-231). In this project, different peptide substrates of EYA will be synthesized by changing variable factors and the correlation of structure to enzyme activity and cell apoptosis will be determined by focusing on the following aims: Aim 1: Design and synthesize self-assembling peptide substrates for EYA enzymes; Aim 2: Determining the efficacy of peptide substrates for inhibiting TNBC in spheroid 3D cell cultures. The correlation between the enzyme kinetic and the activity of nanostructures for targeting EYA will be evaluated. Aim 3: The apoptosis response of the TNBC cells will be determined. This study will lead to finding the potent peptide substrate and the effective dose for inhibiting TNBC cells with apoptosis cell death.