Polo-like kinase 1 (PLK1) is a member Ser/Thr protein kinases that play crucial roles human cell cycle; from controlling mitotic entry, facilitating DNA replication, precise regulation of the cell division and maintenance of genomic stability in mitosis, and DNA damage response (Weerdt et al, Cell cycle, 2006). PLK1 is overexpressed in several human cancers and its knockdown leads to the inhibition of tumour cell proliferation, induction of apoptosis, and increased sensitivity of tumour cells to cancer chemotherapy (Weiß L, Experimental hematology & oncology, 2012). It is a validated chemotherapeutic target and several drugs have been developed as ATP-competitive inhibitors targeting its kinase domain. However, because of resistance and non-selectivity issues associated with the highly conserved nature of ATP binding domain and frequent mutations, the focus has now been moved to the polo-box domain (PBD) of PLK1 (Buschbeck M. Drugs in R&D, 2006). We have found that the cell division cycle-associated protein 3 (CDCA3) mediates cell cycle progression by modulating PLK1 function. The project focuses on the design and development of the protein-protein interaction (PPI) inhibitors of PLK1 and CDCA3, that would provide an alternative treatment modality to cause mitotic arrest in cancerous cells. Herein, we have utilized computational modelling to design the peptide inhibitors based on the interaction between CDCA3 and PLK1-PBD. The designed peptides were evaluated for PLK1 inhibitory activity using a fluorescence polarisation-based biochemical assay and cell-based assays. The identified peptide hit was found to bind to PLK1-PBD at a low-micromolar IC₅₀ value. This peptide also displayed cellular activity in cancer cell lines. The structural information of the active peptide has been used for further optimization of the peptide and development of structure activity relationship. This study demonstrates a novel approach to modulate the PLK1 function via peptides designed from its interactome.