PROteolysis TArgeting Chimeras (PROTACs) are tripartite molecules consisting of a linker connecting a ligand for a protein of interest to an E3 ligase recruiter, whose rationale relies on proteasome-based protein degradation. PROTACs have expanded as a therapeutic strategy to open new avenues for unmet medical needs. Leveraging our expertise, we undertook a series of in vitro experiments aimed at elucidating PROTAC metabolism. In particular, we focused on PROTACs recruiting the von Hippel-Lindau (VHL) E3 ligase. After high-resolution mass spectrometry measurements, a characteristic metabolite with mass reduction of 200 units was detected and successively confirmed as a product deriving from the cleavage of the VHL ligand moiety. Subsequently, we identified hepatic and extrahepatic prolyl endopeptidases as the main putative metabolic enzymes involved. Finally, we designed and synthesized analogs of the VHL ligands that we further exploited for the synthesis of novel VHL-directed PROTACs with an improved metabolic stability in in vitro applications.