A strategy was developed to extend the lifetime of an peptide-based substrate for Abl kinase in the cytosolic environment. Small β-turn structures were added to the peptide's N-terminus to block entry into peptidase catalytic sites. The influence of the size of the β-turn and two covalent cross-linking strategies on the rate of hydrolysis was assessed. The most peptidase-resistant substrate was degraded at a rate of 0.6 pmol mg⁻¹ s⁻¹ and possessed a half-life of 20.3 ± 1.7 min in a Baf/BCR-ABL cytosolic lysate, representing 16- and 40-fold improvements, respectively, over that of a control peptide lacking the β-turn structure. Furthermore, the k_cat/K_M value of this peptide was 432 μM⁻¹ min⁻¹, a 1.25× increase over the unmodified control, verifying that the added β-turn did not hinder the substrate properties of the peptide. This improved peptide was microinjected into single Baf/BCR-ABL cells and substrate phosphorylation measured. Zero to forty percent of the peptide was phosphorylated in the single cells. In contrast, when the control peptide without a β-turn was loaded into cells, the peptide was too rapidly degraded to detect phosphorylation. This work demonstrates that small β-turn structures can render peptides more resistant to hydrolysis while retaining substrate efficacy and shows that these stabilized peptides have the potential to be of high utility in single-cell enzyme assays.