Neurological disorders are the second leading cause of death worldwide. Neurodegenerative diseases such as Alzheimer’s, Parkinson’s, and brain injury are among the most common. These can all result in hypoxic/ischemia conditions affecting catecholamine-producing cells. Tyrosine hydroxylase (TH) is the rate limiting step in catecholamine synthesis. Multiple studies have indicated that the effect of hypoxia causes the increase of TH activity in certain areas of the brain. However, the molecular events and physiological consequences of this process are not fully understood. Hypoxia can be correlated with intracellular acidosis, which alters many cellular processes. In this study, the notion that intracellular acidosis originates from hypoxia and is responsible for the activation of TH is examined. The study uses dopaminergic MN9D cells. Thus, the data suggested that the intracellular acidification of MN9D cells causes unprecedented TH activation through Ser-40 phosphorylation leading to a 7-15-fold over-production of L-3, 4-dihydroxyphenylalanine (DOPA) and dopamine (DA). These findings suggest that the hypoxia/ischemia-induced intracellular acidosis can cause the observed activation of TH, leading to the over-production of DOPA/DA. Further data show that the intracellular acidification-mediated TH activation is associated with the increased intracellular ROS production. Based on several supporting experimental results, we propose that the intracellular acidosis mediated TH activation is correlated with increased ROS production in catecholaminergic cells and contribute to hypoxia and ischemia in the brain. Further studies include confirmation with in vivo studies. It can be purposed that TH could be an effective molecular target for the development of pharmacological agents against neuronal damage from the brain ischemia and/or hypoxia-associated intracellular acidosis.