Encouraged by the performance characteris=cs of iTCO in buffered aqueous systems, we aimed to quan=fy iTCO-release dynamics in real-=me and study its behavior across progressively complex biological systems. Therefore, fluorogenic TCO-caged 7-amino-4-methyl-coumarin (AMC) probes were used, which restore the fluorescence of the free fluorophore upon cleavage and thus provide a convenient real-=me readout (Fig. ). For a direct comparison, we assessed the reac=ons of Pyr2 (1) with iTCO-AMC ( ) and cTCO-AMC (17). For accurate quan=fica=on of the release rate, we selected a Tz concentra=on of 100 µM to facilitate complete click (>99%) in under 5 seconds. The resul=ng release curve recapitulated the HPLC data (cf. Fig. ), independently corrobora=ng the reac=on kine=cs with complete click-to-release in <1 min and helping to rule out any HPLC-related ar=facts, such as instrumental errors and environmental changes introduced during analysis . Fi{ng of the curve resulted in a first-order constant of 0.144 s -1 , which, strikingly, closely aligns with the rate we observed for the omnidirec=onal cleavage of C2TCO in scission-accelerated fluorophore exchange , represen=ng the fastest Tz/TCO elimina=on reac=on at physiological pH. In contrast, only minimal release (<1%) was observed for cTCO over the same =meframe (Fig. ).