A key challenge in Parkinson's disease research is to understand mechanisms underlying selective degeneration of dopaminergic neurons mediated by genetic factors such as α-synuclein (α-Syn). The present study examined whether dopamine (DA)-dependent oxidative stress underlies α-Syn-mediated neurodegeneration using Drosophila primary neuronal cultures. Green fluorescent protein (GFP) was used to identify live dopaminergic neurons in primary cultures prepared on a marked photoetched coverslip, which allowed us to repeatedly access preidentified dopaminergic neurons at different time points in a non-invasive manner. This live tracking of GFP-marked dopaminergic neurons revealed age-dependent neurodegeneration mediated by a mutant human α-Syn (A30P). Degeneration was rescued when α-Syn neuronal cultures were incubated with 1 mm glutathione from Day 3 after culturing. Furthermore, depletion of cytoplasmic DA by 100 µmα-methyl-p-tyrosine completely rescued the early stage of α-Syn-mediated dopaminergic cell loss, demonstrating that DA plays a major role in oxidative stress-dependent neurodegeneration mediated by α-Syn. In contrast, overexpression of a Drosophila tyrosine hydroxylase gene (dTH1) alone caused DA neurodegeneration by enhanced DA synthesis in the cytoplasm. Age-dependent dopaminergic cell loss was comparable in α-Syn vs dTH1-overexpressed neuronal cultures, indicating that increased DA levels in the cytoplasm is a critical change downstream of mutant α-Syn function. Finally, overexpression of a Drosophila vesicular monoamine transporter rescued α-Syn-mediated neurodegeneration through enhanced sequestration of cytoplasmic DA into synaptic vesicles, further indicating that a main cause of selective neurodegeneration is α-Syn-induced disruption of DA homeostasis. All of these results demonstrate that elevated cytoplasmic DA is a main factor underlying the early stage of α-Syn-mediated neurodegeneration.