The macrophage cytotoxic response is dependent on oxygen (O2) as a substrate for oxidant production. Indeed, O2 is utilized by (the inducible) nitric oxide synthase (iNOS) and NADPH oxidase-2 (NOX-2), to produce nitric oxide (●NO) and superoxide (O2 ●–), respectively (Figure 1). Furthermore, the combination reaction between these species leads to peroxynitrite (ONOO–), a strong oxidant that can cause biological damage through oxidation and nitration. Since oxygen concentration varies between tissue and culture conditions, it has been of great interest to study these processes in cellula, considering the physiological environment. Previous reports from our group showed that oxidant production depends on oxygen partial pressure (pO2) when evaluated during short-time exposures (Figure 2). Herein, we show the effects of long-term exposure to a range of physiological pO2 on the activity and expression of iNOS. Peroxynitrite formation, assessed through the boronate-based probe, coumarin-boronic ester (CBE), also shows a similar tendency with pO2 in short-term exposures (scheme 1). Nevertheless, CBE is limited to only certain methodologies because of its spectroscopic properties. Moreover, other probes such as fluorescein-boronate (Fl-B) are sensitive to drastic variations in intracellular pH and pO2. Therefore, we are developing a novel fluorescent boronate-based probe (Red-B) derived from xanthene, to achieve an accurate cellular detection of peroxynitrite in different cellular conditions.