The growing diversity of ABS probes for BL imaging has enabled the study of various important analytes using luciferase-expressing cells and animal models. Most of these examples are prepared by capping the phenolic alcohol of luciferin (or related molecules) with an analyte-responsive trigger, which upon removal can interact with luciferase to yield BL. While luciferin and probes derived from this parent molecule boast excellent BL properties (e.g., strong signal output), the emission wavelength is still in the visible window (λmax = ~560 nm), limiting the attainable imaging depth. A transition to the NIR region (>650 nm) can improve tissue penetration since there are less optical absorbers (e.g., amino acids and nucleic acids) that can intercept, and scatter the emitted light. However, red-shifting luciferase substrates often entails exchanging the alcohol group with a dialkylamino substituent, which prevents installation of triggers at this position. This was in fact the case with NIR emitting BL660, which features a N,Ndiethylamino group. Thus, our only option was to cap the carboxylate with a trigger; however, as discussed previously, the variety of suitable triggers are severely limited. In this work, we overcame this limitation by rationally developing a hydrolysis-resistant ester that is also remarkably stable against enzymatic degradation. By installing a trigger at the para-or ortho-positions of this ester, we can leverage self-immolative chemistry to afford NIR ABS probes via carboxylate-masking and subsequent unmasking. Specifically, inclusion of a nitro group transformed the Ph i Pr ester into an ABS linker for sensing hypoxia in vivo via NTR activity. Our probe, BL660-NTR, is the first NIR BL probe for this target, and only the second NIR emitting ABS probe ever developed. Beyond sensing hypoxia, our new self-immolative linker can be readily adapted to develop probes for other analytes. In addition to modifying BL660, we have also successfully masked methotrexate, which is notorious for severe sideeffects when administered to patients owing to non-specific destruction of healthy cells. Moreover, carboxylates are found in many other medications, including antibacterial agents (ceftazidime, ciprofloxacin, and sulbactam). As such, the pursuit of prodrugs via carboxylate masking using our technology is an exciting avenue.