Human epidermal growth factor receptor-2 (HER2) is a tyrosine kinase family protein receptor that is known to undergo heterodimerization with other members of the family of epidermal growth factor receptors (EGFR) for cell signaling. Overexpression of HER2 and deregulation of signaling has implications in breast, ovarian, and lung cancers. We have designed several peptidomimetics to block the HER2-mediated dimerization, resulting in antiproliferative activity for cancer cells. In this work, we have investigated the structure–activity relationships of peptidomimetic analogs of Compound 5. Compound 5 was conformationally constrained by N- and C-terminal modification and cyclization as well as by substitution with d-amino acids at the N-and C-termini. Among the compounds studied in this work, a peptidomimetic Compound 21 with d-amino acid substitution and its N- and C-termini capped with acetyl and amide functional groups and a reversed sequence compared to that of Compound 5 exhibited better antiproliferative activity in HER2-overexpressed breast, ovarian, and lung cancer cell lines. Compound 21 was further evaluated for its protein–protein interaction (PPI) inhibition ability using enzyme fragment complementation assay, proximity ligation assay, and Western blot analysis. Results suggested that Compound 21 is able to block HER2:HER3 interaction and inhibit phosphorylation of the kinase domain of HER2. The mode of binding of Compound 21 to HER2 protein was modeled using a docking method. Compound 21 seems to bind to domain IV of HER2 near the PPI site of EGFR:HER2, and HER:HER3 and inhibit PPI. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 693–702, 2014.