Prion seed amplification assays, such as real-time quaking-induced conversion (RT-QuIC), are providing ultrasensitive, specific, and increasingly practical means of detecting prions and diagnosing prion diseases in humans and other animals. Widespread efforts have greatly increased the breadth of RT-QuIC applications for research, surveillance, and clinical purposes. Here, we summarize recent progress in several key areas in which RT-QuIC assays are being applied and further developed.