Parkinson’s disease (PD) is characterized by the progressive accumulation of neuronal intracellular aggregates largely composed of alpha-Synuclein (αSyn) protein. The process of αSyn aggregation is induced during aging and enhanced by environmental stresses, such as the exposure to pesticides. Paraquat (PQ) is an herbicide which has been widely used in agriculture and associated with PD. PQ is known to cause an increased oxidative stress in exposed individuals but the consequences of such stress on αSyn conformation remains poorly understood. To study αSyn pathogenic modifications in response to PQ, we exposed Drosophila expressing human αSyn to a chronic PQ protocol. We first showed that PQ exposure and αSyn expression synergistically induced fly mortality. The exposure to PQ was also associated with increased levels of total and phosphorylated forms of αSyn in the Drosophila brain. Interestingly, PQ increased the detection of soluble αSyn in highly denaturating buffer but did not increase αSyn resistance to proteinase K digestion. These results suggest that PQ induces the accumulation of toxic soluble and misfolded forms of αSyn but that these toxic forms do not form fibrils or aggregates that are detected by the proteinase K assay. Collectively, our results demonstrate that Drosophila can be used to study the effect of PQ or other environmental neurotoxins on αSyn driven pathology.