Transla=ng these findings into a cellular context, we inves=gated the bioorthogonal ac=va=on of the iTCOprodrugs in HT1080 fibrosarcoma cells (Fig. ). In alignment with the HPLC data, the in situ reac=on of the iTCO-caged drugs with both Pyr2 (15) and MeBAm (13) fully restored the cytotoxicity of the free parent drugs. Notably, these cell experiments, like most widely used cell viability assays, omit washing steps, resul=ng in the reac=on occurring predominantly in the extracellular space-i.e., in the surrounding media, rather than inside cells. An assay specific for intracellular click-to-release demands an experimental strategy for enrichment of Tz within cells and removal of extracellular Tz, while imposing cell-specific limits on stoichiometry and concentra=on. The addi=onal molecular complexity of the cytoplasmic environment offers a further challenge to assess the biodistribu=on, stability and capability of the tools. To this end, we func=onalized MeBAm with a triphenyl-phosphonium (TPP) moiety (Fig. ), enabling selec=ve, chargedriven mitochondrial localiza=on, as previously demonstrated . HT1080 cells were incubated with MeBAm-TPP (18, 10 µM, 2 h) to achieve selec=ve pretarge=ng to mitochondria, followed by washing to remove extracellular Tz. Subsequent treatment with iTCO-CA4 at low nanomolar concentra=ons (20 nM) triggered potent cytotoxicity (Fig. ). Pretargeted prodrug ac=va=on across different iTCO-CA4 concentra=ons revealed a dose-response rela=onship with an IC50 of 3.3 nM, matching that of the parent drug CA4 (see Supplementary Informa=on). This confirms efficient intracellular ac=va=on via click-to-release and highlights the bioorthogonal performance of the system, including cell permeability and iTCO stability.