Peptoids are an emerging class of peptidomimetics that received much attention in recent times due to their drug-like properties. In comparison to conventional peptide and antibody therapeutics, peptoids exhibit improved serum and chemical stability, tissue and cell permeability, and reduced immunogenicity. The economical and straightforward synthesis of peptoids allows the development of peptoid libraries with millions of diversities. These bead-displayed peptoid libraries can be synthesized very rapidly (within 3-5 days) and can be utilized in high throughput screening in the modern drug discovery process. Our project was initiated to identify “true” early markers for non-small cell lung cancer (NSCLC) and specific chemical ligands such as peptoids to target them. Our collaborators successfully developed a model of in vitro malignant transformation of normal bronchial cells to full malignancy via the stepwise introduction of key oncogenic mutations such as alternations of p53, KRAS, and cMyc. We hypothesized that novel cancer-specific cell surface markers may appear due to these oncogenic alternations, and then those can be captured by bead-displayed peptoids. Therefore, we exposed bead-displayed peptoid libraries to those oncogenically transformed cells to capture potential new markers through peptoid ‘hits’ using our unique on-bead two-color (OBTC) screening method, which compares this recognition to the normal bronchial cells in real-time and avoid peptoids binding to common markers. One of the highly selective peptoids ‘hits’ identified was named as JM3A and the targeted new marker was identified and validated as cell surface vimentin (CSV). Vimentin is a structural protein universally found in the cytosol but uniquely translocated to the cell surface in metastatic cancer stem cells (CSCs). Our first approach is to optimize the binding of peptoid JM3A on CSV using various medicinal chemistry approaches and extract the antagonistic (anti-cancer) effect. We first performed minimum pharmacophore identification studies using alanine/sarcosine scans. These studies revealed that residues 1–4 and 8 (from the C-terminus) are not important and those residues 5–7 are important for the direct binding of JM3A to CSV. We then found that our previous N-terminal benzophenone(BP)-coupled JM3A (JM3A-BP), which was used for pull-down and target identification studies, displayed 3-fold binding enhancement. Molecular docking studies indicated that the BP moiety binds to a binding pocket on the vimentin coil 2 fragment, and further studies using 12 benzophenone-like moieties indicated that at least two phenyl groups are needed to occupy this binding pocket. Interestingly, the binding was improved when non-important and bulky residues at the 4th and 8th positions were replaced with methyl groups (JM3A-4,8-BP). We next dimerized JM3A-4,8-BP to enhance the binding via the avidity effect, using a central lysine linker to develop JM3A-4,8-BPD1 (EC50 = 300 nM). This showed 27- and 63-fold-improvement in binding over JM3A-4,8-BP and JM3A monomers, respectively. JM3A4,8BPD1 also displayed binding comparable to commercially available vimentin antibody. Finally, we observed an antagonist effect on H1299 NSCLC cell proliferation and viability from this optimized dimeric JM3A-4,8BPD1, which was not shown by the monomeric derivatives. Next, we focused on optimizing the linker length between two JM3A-BP monomers targeting CSV to allow two monomeric units to precisely bind their counterparts and hence further enhance binding and anti-cancer activity. First, seven JM3A derivatives with varying linker lengths/types were synthesized and evaluated by the standard MTS cell viability assay for anti-cancer activity. The most optimized derivative contained a central lysine linker and two glycines, named L2 with Kd ~ 100 nM and IC50~ 6.7 µM. Next, the methionine at N-terminus was replaced with D-alanine (JM3A-L2Dala) to enhance the yield and stability. The JM3AL2-Dala exhibited a similar activity as L2 and displayed 10-fold and 180-fold improvements in binding affinity compared to JM3A-BP dimer and original screening hit JM3A, respectively.JM3A-L2Dala was later tested on different NSCLC cells with different vimentin expression levels and results suggested that JM3A-L2Dala exhibited more potency on high vimentin-expressing cells (H1299, H460) compared to low vimentin-expressing cells (H2122). No activity was observed on JM3AL2-Dala-treated normal bronchial HBEC-3KT cells. Assessment of the inhibitory activity of stemness by JM3A-L2Dala on cancer stem cells (CSCs) was performed by using the standard colony formation assay and wound healing assay. The result indicated that JM3A-L2Dala can disrupt colony formation starting from 400 nM and inhibit cell migration activity at 2 µM. The mechanism of action studies suggested that L2-Dala induced apoptosis via a dose-and time-dependent manner. We envision that further optimization of our vimentin binding peptoid JM3A can be utilized in future anti-CSCs drug discovery as well as early NSCLC detection strategy developments.