Isolated peptide substrates of Plk1 and nucleic acids encoding these peptides are disclosed. The peptides include two to ten repeats of the amino acid sequence set forth as XXAXXXPLHSTXXXXXXX(SEQ ID NO: 1), in which within each repeat X, X, X, X, X, X, X, X, and Xare each independently any amino acid or no amino acid, and Xand Xare each independently any amino acid. Methods of using these peptides to detect Plk1 activity in a sample are also disclosed. In some examples, the method includes contacting a sample with a disclosed peptide substrate of Plk1 in the presence of adenosine triphosphate, or an analog thereof, for a period of time sufficient for Plk1 to phosphorylate the PBIPtide. The presence and/or amount of the phosphorylated and/or the unphosphorylated peptide is detected, thereby detecting and/or quantitating Plk1 kinase activity in the sample.