Fluorescence polarization (FP) is a reliable and straightforward mix-and-read assay to quantify the interaction of a protein–peptide pair. Labeling the peptide with a suitable fluorophore enables the distinction between the protein-bound and protein-unbound states of a peptide by a change in the emitted light’s polarization. Hereby, we present the method to establish the binding affinity for FOXO3a phosphopeptide with 14-3-3ɛ protein using FP. With the advantages of lower sample consumption and faster read-outs, FP validates the interaction between the pairs and can also be used to develop a competitive assay to identify and validate the novel inhibitors for the interaction pair.