The uptake and accumulation of pathological α-synuclein aggregates in neuronal cells is thought to be a major underlying mechanism in the pathogenesis of Parkinson’s disease. This protocol describes an assay to quantify the uptake of these aggregates by neuronal cells by monitoring the uptake of fluorescently labelled α-synuclein preformed fibrils (PFFs) in dopaminergic (DA) neural progenitor cells (NPCs) derived from induced pluripotent stem cells (iPSCs). This protocol provides the details and methods we used for other researchers to screen small molecule libraries for actives that modulate the uptake of fluorescently labelled PFFs uptake in NPCs. It describes the assay, subsequent image acquisition, and image analysis by high-content screening (HCS).