This chapter discusses pseudosubstrate-based peptide inhibitors. Many protein kinases exist as catalytically inactive or latent forms unless activated by the binding of a ligand or by covalent modification, such as phosphorylation. The inactive form of protein kinases results from the presence of an autoinhibitory region that masks the catalytic activity. Subsequent removal of the pseudosubstrate region from the active site is required for expression of protein kinase activity. Pseudosubstrate-based peptide inhibitors can be used to study the physiological roles of their respective protein kinases in extracts or in cells by microinjection, use of permeabilized cell, or expression in transfected cells. There may be overlaps in the specificity of pseudosubstrate peptides. Therefore, it is necessary to include several types of controls in such studies. These include an inactive pseudosubstrate analog peptide as a negative control and, if available, the constitutively active form of the protein kinase as a positive control.