The cellular prion protein, PrPC, is attached by a glycosylphosphatidylinositol anchor to the outer leaflet of the plasma membrane. Its misfolded isoform PrPSc is the causative agent of prion diseases. Conversion of PrPC into PrPSc is thought to take place at the cell surface or in endo-lysosomal organelles. Understanding the intracellular trafficking of PrPC may therefore help elucidating the conversion process. Here we describe a time-resolved fluorescence resonance energy transfer (FRET) assay reporting membrane expression and real-time internalization rates of PrPC The assay is suitable for high-throughput genetic and pharmaceutical screens for modulators of PrPC trafficking. Simultaneous administration of FRET donor and acceptor anti-PrPC antibodies to living cells yielded a measure of PrPC surface density, whereas sequential addition of each antibody visualized the internalization rate of PrPC (Z'-factor > 0.5). RNA interference assays showed that suppression of AP2M1, RAB5A, VPS35 and M6PR blocked PrPC internalization, whereas downregulation of GIT2 and VPS28 increased it. PrPC cell surface expression was reduced by downregulation of RAB5A, VPS28 and VPS35, and enhanced by silencing EHD1. These data identify a network of proteins implicated in PrPC trafficking, and demonstrates the power of this assay for identifying modulators of PrPC trafficking.