Aim: The conjugation of fluorescent labels to opioid peptides is an extremely challenging task, which needs to be overcome to create new classes of probes for biological assays. Materials & methods: Three opioid peptide analogs of biphalin and [D-Pen2,5]-Enkephalin (DPDPE) containing a fluorescein–maleimide motif were synthesized. Results & discussion: The biphalin analog 17 binds to opioid receptors with K_i ^μ = 530 ± 90 nM and K_i ^δ = 69.8 ± 16.4 nM. We then tested the ability of the compounds to stimulate G-protein-coupling, 17 activated μ-receptor expressing cells (EC₅₀ = 16.7 ± 6.7 nM, E_Max = 76 ± 4%) as well as δ-receptor expressing cells (EC₅₀ = 42 ± 10 nM, E_Max = 34 ± 8%). However, 17 was not able to fluorescently label receptor in live or fixed cells. Conclusion: Our data suggest that the biphalin scaffold could be employed to develop fluorescent ligands with the appropriate fluorescent motif, and suggest a means for further probe development.