Enzymatic activity sensing in fluorescence lifetime (FLT) mode with “self-quenched” macromolecular near-infrared (NIR) sensors is a highly promising strategy for in vivo imaging of proteolysis. However, the mechanisms of FLT changes in such substrate-based NIR sensors have not yet been studied. We synthesized two types of sensors by linking the near-infrared fluorophore IRDye 800CW to macromolecular graft copolymers of methoxy polyethylene glycol and polylysine (MPEG-gPLL) with varying degrees of MPEGylation and studied their fragmentation induced by trypsin, elastase, plasmin, and cathepsins (B,S,L,K). We determined that the efficiency of such NIR sensors in FLT mode depends on sensor composition. While MPEG-gPLL with a high degree of MPEGylation showed rapid (τ1/2 = 0.1–0.2 min) FLT increase (Δτ = 0.25 ns) upon model proteinase-mediated hydrolysis in vivo, lower MPEGylation density resulted in no such FLT increase. Temperature-dependence of fluorescence dequenching of NIR sensors pointed to a mixed dynamic/static-quenching mode of MPEG-gPLL-linked fluorophores. We further demonstrated that although the bulk of sensor-linked fluorophores were dequenched due to the elimination of static quenching, proteolysis-mediated deletion of a fraction of short (8–10 kD) negatively charged fragments of highly MPEGylated NIR sensor is the most likely event leading to a rapid FLT increase phenomenon in quenched NIR sensors. Therefore, the optimization of “built-in” dynamic quenching elements of macromolecular NIR sensors is a potential avenue for improving their response in FLT mode.