The development and application of tools that allow labeling of proteins in vitro and in vivo is a highly active and interdisciplinary area of research. Self-immolative polymers (SIPs) are a unique type of molecules that respond to external stimuli by undergoing head-to-tail disassembly through a self-immolative fragmentation. We have demonstrated how these polymers can be applied as activity-linked labeling probes for proteins with catalytic activity. A real-time detection of the protein labeling can be monitored due to a change in the fluorescence emission wavelength of the probe during the polymer disassembly process. The SIP-based labeling approach showed considerable conservation of catalytic activity of the labeled protein and significant preference toward labeling of the activating protein.