Given its high metabolism, the brain is especially prone to injury from mitochondrial dysfunction. Diffuse optical spectroscopy (DOS) provides a non-invasive means to monitor mitochondrial metabolism. Accordingly, DOS can improve understanding of the pathophysiology of mitochondrial dysfunction. DOS assesses metabolism with both mitochondrial and hemodynamic metrics. The mitochondrial metric probes the change in redox state of cytochrome-c-oxidase (or complex IV), while the hemodynamic metrics include cerebral blood flow (CBF) and the oxygen extraction fraction (OEF) of cerebral tissue. Prior work with these metrics has largely focused on the optical detection of impaired metabolism caused by hypoxia and low blood flow (ischemia). These are important causes of metabolic failure, but other failure causes exist. Indeed, mitochondrial dysfunction commonly occurs during non-ischemic conditions in patients with Parkinson’s, Alzheimer’s, and other diseases.In my thesis research, I used DOS to study dysfunction arising from mitochondrial complex I impairment. To my knowledge, this is the first use of DOS to study mitochondrial impairment that isn’t caused by ischemia or hypoxia. The study was performed in a swine model of complex I impairment induced by rotenone exposure. I showed that the DOS mitochondrial metric, but not the OEF metric, was sensitive to the mild impairment. In addition, I used DOS to monitor the efficacy of a novel therapeutic of mitochondrial dysfunction, i.e., NV354 succinate prodrug. DOS measurements supported NV354’s ability to increase cerebral oxygen metabolism in this model. In another novel contribution, I used DOS to study cerebral metabolism during carbon monoxide (CO) poisoning in swine. CO can impair metabolism via its binding to mitochondrial complex IV, and via its decrease in arterial oxygen saturation (SaO2). Swine were exposed to moderate (1000 ppm) or severe (2000 ppm) CO levels for two hours. DOS showed evidence of metabolic impairment in the swine with severe CO poisoning, but not in the swine with moderate CO poisoning. Systemic arterial oxygen saturation, in contrast, was diminished in both the moderate and severe groups. DOS thus promises to enhance sensitivity to cerebral injury. Together, these studies motivate DOS’ utility in managing patients with mitochondrial impairment that isn’t caused by cerebral ischemia/hypoxia.