Overproduction of reactive oxygen species (ROS) can lead to several disease states, such as diabetic nephropathy and amyotrophic lateral sclerosis. One of the most studied mechanisms to inhibit the over production of ROS is the inhibition of NADPH oxidase (NOX) enzymes, which catalyze the conversion of cytoplasmic NADPH to NADP, resulting in the formation of superoxide anions. GSK2795039 has been shown to selectively inhibit the NOX2 isoform, however, clearance of the compound was high in rats and mice. Therefore, identifying metabolic soft spots would be crucial in guiding the optimization process to improve its pharmacokinetic properties. GSK2795039 (10 μM) was incubated in the presence of mouse, rat and human liver microsomal (1 mg/mL) and cytosolic (2 mg/mL) fractions and appropriate co-factors, followed by MS fragment analysis to identify metabolic soft spots. GSK2795039 showed marked species differences in its metabolism. The alkyl side chains and indoline moiety were the most common sites of biotransformation. The compound was identified to be an aldehyde oxidase substrate. Additionally, unique human metabolites were observed in vitro. Our study sheds light on structure optimization opportunities for developing improved NOX2 inhibitors, and it will help overcome the challenges involved in preclinical species selection for its safety evaluations.