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Polo-like kinase (Plk) plays a central role in centrosome cycle and is closely associated with the oncogenesis of lung cancer. The protein consists of a catalytic kinase domain (KD) and a regulatory polo-box domain (PBD); either direct inhibition of the KD's catalytic activity or indirect disruption of the PBD-substrate interaction can be used to potentially suppress the pathological activation of lung cancer Plk. Here, we reported a successful molecular design and engineering of phosphopeptide ligands to target Plk PBD domain by integrating in silico modeling and in vitro assay. In the procedure, a helical peptide segment hps was derived from dimerization interface of the complex crystal structure of domain dimer using bioinformatics approach, which was then used as sequence template to generate potent phosphopeptide binders of Plk PBD domain in terms of a systematic residue mutation profile. Fluorescence anisotropy assays were conducted to substantiate the findings and conclusions obtaining from the molecular engineering. Consequently, three helical phosphopeptides, including the native hps and its two mutants hps-m and hps-m , were successfully designed that can independently rebind to Plk PBD domain with a moderate or high affinity ( K = 127, 26, and 5 μM, respectively). These peptide ligands can be considered as potent self-competitors to disrupt PBD dimerization in lung cancer metastasis. Structural and energetic analysis revealed that hydrophobic forces and van der Waals contacts confer strong stability for domain-peptide complex system, while hydrogen bonds and electrostatic interactions contribute specificity and selectivity to the complex recognition. [ABSTRACT FROM AUTHOR]