Metal-ion responsive fluorescent probes are powerful tools for the non-invasive visualization of metal ion activities in biology. Given the tight attomolar buffering of kinetically labile Cu(I) in mammalian cells,1 the in situ detection of cellular Cu(I) poses significant challenges. Reaction-based probes utilizing tris-picolyl amine (TPA) for the Cu(I)-mediated oxidative cleavage and fluorescence activation have emerged as an alternative approach to traditional fluorescence sensing schemes that rely on reversible Cu(I) coordination. However, TPA is also an effective cellular Zn(II) chelator,2 and its Cu(I) affinity is several orders of magnitude weaker3 compared to glutathione4 and proteins engaged in Cu(I) trafficking, thus raising the question to what extent indirect or Cu(I)-independent redox processes might contribute to the fluorescence response in live cells. To elucidate the role of such redox processes in fluorescence activation, we tethered TPA to three structurally distinct fluorophore platforms. Detailed kinetic and spectroscopic studies revealed that ternary complex formation with GSH-bound Cu(I) was essential for rapid oxidative cleavage of the probes and independent of the nature of the fluorophore. Moreover, TPA-Cu(I)-induced superoxide production and reaction with diffusible superoxide were orders of magnitude slower. Likewise, the fluorogenic response was abrogated in the presence of MCL-2,5 a chelator with higher Cu(I) affinity than TPA but lower affinity than GSH, thus underscoring the role of GSH for the Cu(I)-induced oxidative cleavage of the probes. Given the absence of a sizeable pool of GSH-bound Cu(I) under normal physiological conditions,1 the fluorescence activation of TPA-based probes might involve Cu(I)-independent enzymatic pathways. Altogether, the complexity of the cellular environment underscores the challenges when interpreting the fluorescence response of irreversible reaction-based synthetic probes.