Activity-based sensing (ABS) probes equipped with a NIR bioluminescence (BL) readout are promising chemical tools to study cancer biomarkers owing to their high sensitivity and deep tissue compatibility. However, the standard approach of installing a responsive trigger at the aniline site through a self-immolative linker is not suitable for NIR substrates because they require N,N-dialkylation at this position to achieve NIR emission. Capping the carboxylate is also unfavorable due to the instability of the resulting ester moiety which would result in high background signals. In this study, we rationally designed a hydrolysis-resistant ester featuring an isopropyl shielding arm. Compared to a benzyl ester analog (proxy for self-immolative linker), the new design is 140.5-fold and 67.8-fold more resistant toward spontaneous and esterase-mediated hydrolysis, respectively. After further in cellulo evaluation of stability, this ester moiety was transformed into a general self-immolative linker for ABS probe development via carboxylate masking. We showcased the utility of this technology by developing the first NIR BL probe for hypoxia sensing (BL660-NTR) and applied it in lung cancer cells and in a murine model of non-small cell lung cancer.