The misfolding of proteins such as the prion protein, α-synuclein, and tau represents a key initiating event for pathogenesis of most common neurodegenerative disorders, and its presence correlates with infectivity. To date, the diagnosis of these disorders mainly relied on the recognition of clinical symptoms when neurodegeneration was already at an advanced phase. In recent years, several efforts have been made to develop new diagnostic tools for the early diagnosis of prion diseases. The real-time quaking-induced conversion (RT–QuIC) assay, an in vitro assay that can indirectly detect very low amounts of PrPSc aggregates, has provided a very promising tool to improve the early diagnosis of human prion diseases. Over the decade since RT–QuIC was introduced, the diagnosis of not only prion diseases but also synucleinopathies and tauopathies has greatly improved. Therefore, in our study, we summarize the current trends and knowledge of RT–QuIC assays, as well as discuss the diagnosis of neurodegenerative diseases using RT–QuIC assays, which have been updated in recent years.