The bioavailability and cell permeability of small-molecule drugs, imaging agents, or activity-based sensing (ABS) probes are largely dictated by molecular weight, lipophilicity, hydrogen bonding ability and net charge. For instance, a positively charged or neutral molecule is more likely to gain entry into a cell compared to an anion owing to Coulombic repulsion caused by the negatively charged membrane. A reliable strategy to improve cell uptake is to temporarily mask the negative functional group. For example, carboxylates can be readily transformed into the corresponding ester moiety, which upon crossing the cell membrane, can be cleaved to unmask the latent charge via intracellular esterase activity. A notable example is the use of this strategy to facilitate uptake of fluorescent indicators for calcium, which are equipped with a tri-or tetra-carboxylate binding motif. Under normal circumstances, it would be nearly impossible for a molecule with a net charge of -3 or -4 to enter the cell. However, calcium indicators for cellular imaging are routinely prepared and sold in the acetoxy methyl ester form. Beyond the modification of small molecules, this approach has extended to facilitate the delivery of functional proteins through the conversion of surface-exposed carboxylates to esters. Elegant examples include the delivery of GFP and human ribonuclease 1. However, there are limitations that must be considered when carboxylate-masking is employed in vivo for the purpose of targeted cargo delivery. First, most esters are prone to spontaneous hydrolysis under acidic, neutral, or basic conditions. The body features numerous pH gradients (e.g., the pH in the gastrointestinal tract ranges from 5.7 to 7.4), which will result in off-target delivery upon systemic administration. Second, ester-hydrolyzing enzymes are ubiquitously expressed throughout the body in nearly all cell types, leading to indiscriminate cargo release. In general, ester stability is correlated to size and this property has been leveraged to tune the pharmacokinetics of drugs such as androgens. For instance, the in vivo half-life of testosterone esters can be increased from the 2-3-day range (propionate ester) to over 12 weeks (undeconate ester) by simply varying the length of the alkyl chain. In the context of ABS, an assortment of ester-based probes exists; however, an important distinction is that the carboxylate is a component of the trigger and not of the reporter. Amongst the ABS probes designed for in vivo applications, those equipped with a bioluminescence (BL) readout are highly coveted. Unlike fluorescence-based probes which require external light excitation to generate a signal, BL results from luciferase-mediated light production and thus, is highly sensitive due to low background. Moreover, the structure of luciferase substrates can be modified to shift the emission wavelength into the NIR, as we have done recently, to achieve greater tissue penetration. Regardless of the variant, a viable substrate must contain a free carboxylate group for enzyme recognition and subsequent light generation. Unfortunately, the presence of a negative charge adversely impacts cell permeability. To yield an appreciable BL signal in vivo, luciferin must be administered systemically at exorbitant levels (>150 mg/kg). Recently, we reported the development of BL660-NO (a nitric oxide (NO)-responsive probe) by amidating the carboxylate of BL660 (a NIR BL substrate) with O-phenylene diamine. We found that this modification dramatically improved cell permeability (in vivo dosing decreased to 1 mg/kg), prompting us to explore the installation of other triggers at this position. However, upon surveying the literature, we discovered the available options were limited to hydrolytically stable groups (e.g., amides, aldehydes, etc.), with a few examples of simple esters. We hypothesize the apparent void in this research space is likely due to the general instability of esters in vivo (Scheme 1). In this study, we report the successful development of a new hydrolysis-resistant ester. Moreover, we show that carboxylate-masking using this ester is generalizable through the modification of two sets of molecules, BL660 and methotrexate (a potent anticancer and immunosuppressive drug). Importantly, we converted the optimized ester to a new self-immolative linker, which we used to develop an esterbased probe to sense tumor hypoxia. The utility of the resulting probe (BL660-NTR) was showcased in A549 lung cancer cells, as well as in a murine model of lung cancer via NIR BL imaging.