Hydroxylation and fluorination of proline alters the pyrrolidine ring pucker and the trans:cis amide bond ratio in a stereochemistry-dependent fashion, affecting molecular recognition of proline-containing molecules by biological systems. While hydroxyprolines and fluoroprolines are common motifs in medicinal and biological chemistry, the synthesis and molecular proper-ties of prolines containing both modifications i.e. fluoro-hydroxyprolines have not been described. Here we present a practical syn-thesis of all four diasteroisomers of 3-fluoro-4-hydroxyprolines (F-Hyps). Conformational preferences and the effect of fluorination on hydrogen bond donating capacity are elucidated by means of X-ray crystallography, NMR spectroscopy and quantum mechani-cal calculations. We exemplify an application of F-Hyps as novel building blocks for biomolecular recognition by incorporating them in HIF-1α peptides and small peptidomimetic ligands targeting the von Hippel-Lindau E3 ligase (VHL) – widely used as PROTAC (PROteolysis TArgeting Chimera) conjugates for targeted protein degradation. We found that VHL exhibits stereoselec-tive recognition of the (3R,4S)-F-Hyp over the corresponding (3S,4S) epimer and rationalize this preference through co-crystal structures and electrostatic potential calculations. Hyp substitution with (3R,4S)-F-Hyp into the BET degrader MZ1 retained bind-ing affinities and cellular degradation activities comparable to those of the parent PROTAC. In spite of a ~20-fold loss in binding affinity to VHL, incorporation of the (3S,4S) epimer into MZ1 led to Brd4-selective degradation at nanomolar concentration. We anticipate that the disclosed chemistry of 3-fluoro-4-hydroxyprolines and their application as VHL ligands for targeted protein deg-radation will be of wide interest to medicinal organic chemists, chemical biologists and drug discoverers alike.