Protein degradation is one of the tactics used by the cell for irreversibly inactivating proteins. In eukaryotes, ATP-dependent protein degradation in the cytoplasm and nucleus is carried out by the 26S proteasome. Most proteins are targeted to the 26S proteasome by covalent attachment of a multiubiquitin chain. A key component of the enzyme cascade that results in attachment of the multiubiquitin chain to the target or labile protein is the ubiquitin ligase that controls the specificity of the ubiquitination reaction. Defects in ubiquitin-dependent proteolysis have been shown to result in a variety of human diseases, including cancer, neurodegenerative diseases, and metabolic disorders. The SCF (Skp1-Cullin-F-box-Hrt1) complex is a heteromeric ubiquitin ligase that multiubiquitinates proteins important for signal transduction and cell cycle progression. A technology was developed known as Protac (Proteolysis Targeting Chimeric Molecule) that acts as a bridge, bringing together the SCF ubiquitin ligase with a protein target, resulting in its ubiquitination and degradation. The Protac contains an SCF-binding peptide moiety at one end that is recognized by SCF that is chemically linked to the binding partner or ligand of the target protein. The first demonstration of the efficacy of Protac technology was the successful recruitment, ubiquitination, and degradation of the protein methionine aminopeptidase-2 (MetAP-2) through a covalent interaction between MetAP-2 and Protac. Subsequently, we demonstrated that Protacs could effectively ubiquitinate and degrade cancer-promoting proteins (estrogen and androgen receptors) through noncovalent interactions in vitro and in cells. Finally, cell-permeable Protacs can also promote the degradation of proteins in cells. This chapter includes experiments to test the ability of Protacs to target proteins in vitro and in cells.