Extracellular cathepsin B is a driver of tumor progression and metastasis, and its potential as a diagnostic and prognostic marker is increasingly recognized. To harness its activity for triggering the uptake of activatable cell-penetrating peptides (ACPPs) in vivo, kinetically suitable and stable endopeptidase substrates for this cysteine protease, which mainly acts as a carboxydipeptidase, are required. This challenge was tackled by C-terminal elongation of the previously identified GIVRAK sequence to octapeptides and systematic structural variation, which has revealed that the endopeptidase activity of cathepsin B is associated with kinetic hysteresis and the P4′ residue plays a key role in this regard, as further investigated by enzyme–substrate docking in silico. By replacing the N-terminal motif with GFLG and focused N-methylation of the backbone, the substrate serum half-life was extended from 3.7 min to 23.4 h. Integrating this sequence into the fluorophore-conjugated ACPP and fluorescence microscopy in U87MG cells confirmed cathepsin B-mediated uptake on the basis of selective inhibitors and control probes. PET imaging and biodistribution studies ex vivo with a NODAGA-conjugated ACPP analogue radiolabeled with copper-64 in a murine U87MG-derived xenograft model together with radiopharmacological investigations in normal Wistar rats demonstrated more favorable pharmacokinetics compared to the corresponding CPP. Although tumor-associated proteolytic activation in vivo is indicated, this does not contribute to tumor retention as judged from control experiments under pharmacological blockade of cathepsin B and with nonfunctional analogues. The obtained results are discussed in the context of previous data for radiolabeled ACPPs, and limitations for the general use of ACPPs for radiotheranostic approaches are highlighted.