In Parkinson's disease (PD), alpha-synuclein (α-syn) aggregation causes neuronal dysfunction and death, particularly of dopaminergic neurons, which is central to PD symptoms and progression. Here, we present a protocol for using a sandwich ELISA to quantify total α-syn levels in various Drosophila melanogaster genotypes expressing human α-syn point mutations associated with PD. We describe steps for aging flies, collecting fly heads, and preparing samples. We then detail procedures for ELISA setup and microplate reader analysis.