Herein, we describe a catalyst-free thia-Diels-Alder cycloaddition for the chemoselective labeling of fully deprotected phosphonodithioester-peptides in solution with fluorophores functionalized with an exocyclic diene. The reaction was optimized on the model tripeptide 1 containing a lysine residue, which enabled its rapid and straightforward labeling with three different fluorophores (fluorescein, lissamine rhodamine B, and squaraine) in very mild conditions (H2O/iPrOH, 37 °C, 1 h). The reaction was then successfully applied to the chemoselective labeling of fully deprotected apelin-13 with squaraine dye. The resulting fluorescent ligand 18 exhibited a high affinity (0.17 ± 0.03 nM) for apelinR. It enabled the development of time-resolved FRET-based competition assays for high-throughput screening and drug discovery. Thanks to its fluorogenic properties, ligand 18 was also successfully involved in the live-cell optical imaging of apelinR in no-wash conditions.