Multiple myeloma (MM) is an incurable malignancy of the antibody-producing plasma cells in the bone marrow and is characterized by the targeted degradation of antitumor regulatory proteins by the ubiquitin proteasome system (UPS). The development of proteasomal inhibitors, such as bortezomib (Velcade®), has greatly improved the median survival time of MM patients. Inappropriate activation of protein kinase B (PKB or Akt) by bortezomib has been observed leading to refractory disease. Inhibitors of PKB activity are now in clinical trials for the treatment of MM. Chronic myelogenous leukemia (CML), the malignancy of myeloid cells in the bone marrow, is treated predominantly with Abl kinase inhibitors. We have designed protease-resistant fluorescent peptide probes that, once optimized, can be used to determine the effectiveness of these inhibitors by direct measurement of proteasomal and Abl kinase hyperactivity and PKB activation in patient cells. Measuring intracellular UPS and kinase activity through the use of unstructured synthetic peptide substrates is limited by the vulnerability of these substrates to rapid degradation in the cell by cytosolic proteases. Unstructured peptides in the cytosol of immune cells are threaded into protease active sites at the N-terminus. We designed and synthesized short, well-folded protease resistant β-hairpin peptides through the incorporation of favorable cross-strand side chain interactions and a stabilizing turn nucleating sequence. Designing the β-hairpins to include ornithine, a noncanonical amino acid analog of lysine, and D-amino acids, produced long-lived peptides with half-lives of 2-6 hours and non-degradable peptides, in the presence of specific and nonspecific proteases in vitro. These β-hairpin peptides were covalently attached to the N-terminus of unstructured kinase substrate peptides resulting in increased stability to peptidases in vitro while maintaining efficacy as kinase substrates. Here we also show that short β-hairpin peptides, not derived from natural protein targets, adequately served as substrates of the UPS. We demonstrated that ornithine can be ubiquitinated by UPS enzymes regardless of secondary structure in a site-specific manner. Finally we show the applicability of these hairpin degrons in determining UPS activity in diseases such as MM, where UPS inhibitors are part of mainline and emerging treatments.