Prion diseases are fatal, progressive, transmissible neurodegenerative conditions that affect both humans and animals. The scrapie prion protein (PrPSc), a misfolded form of prion, is both the cause of prion diseases and gold standard biomarker for diagnosis. Given the infectivity and 100% lethality of prions, new detection methods are urgently needed to enhance the diagnosis and surveillance of prion diseases. In this review, we summarize recent advancements in PrPSc detection. We explore in vitro seeding assays, such as Protein Misfolding Cyclic Amplification and Real-Time Quaking-Induced Conversion, alongside innovative biosensors and imaging techniques developed for prion detection. This review highlights the evolving landscape of PrPSc detection methods and their potential roles in early diagnosis, prion transmission control, and effective treatment development.