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Drug discovery and therapeutic development for disorders of the central nervous system (CNS) represents one of the largest unmet markets in modern medicine. We have increasingly recognized that the lack of stringent assessment of mitochondrial function during the discovery process has resulted in drug recalls, black box warnings, and an urgent need to understand the metabolic liability of small molecules in neural systems. Given that the brain is the most energetically demanding organ, even modest perturbations in neuronal energetic pathways have been shown to impact growth, signaling, connectivity, and the restorative capacity of the CNS. In this work, we describe several tools to assess metabolic activity of primary neuronal cultures and neural cell lines using an acute model of injury induced by oxygen glucose deprivation. Methods include the measurement of total ATP and NADH, enzymatic assessment of lactate production by anaerobic respiration, as well as viability assays. We also present a modified screening method for assessing aerobic respiration of immortalized cell lines using galactose challenge.