G protein‐coupled receptor kinases (GRKs) control the signaling and activation of G protein‐coupled receptors through phosphorylation. In this study, consensus substrate motifs for GRK2 were identified from the sequences of GRK2 protein substrates, and 17 candidate peptides were synthesized to identify peptide substrates with high affinity for GRK2. GRK2 appears to require an acidic amino acid at the −2, −3, or −4 positions and its consensus phosphorylation site motifs were identified as (D/E)X1–3( S / T ), (D/E)X1–3( S / T )(D/E), or (D/E)X0–2(D/E)( S / T ). Among the 17 peptide substrates examined, a 13‐amino‐acid peptide fragment of β‐tubulin (DEMEF T EAESNMN) showed the highest affinity for GRK2 (Km , 33.9 μM; Vmax , 0.35 pmol min−1 mg−1), but very low affinity for GRK5. This peptide may be a useful tool for investigating cellular signaling pathways regulated by GRK2.