BACKGROUND: The diagnosis of neurodegenerative diseases (NDDs), such as Alzheimer disease (AD), Creutzfeldt-Jakob disease (CJD), Parkinson disease (PD), and other α-synucleinopathies, is traditionally based on clinical signs and symptoms. By the time symptoms emerge, neurodegeneration has already advanced, limiting the window for early treatment and research. Early diagnosis could improve therapeutic outcomes and deepen insights into disease onset, making the search for novel diagnostic tools and early biomarkers a major focus of current research. A key pathological hallmark of these prion-like diseases is the misfolding and aggregation of proteins such as prion protein, α-synuclein (αSyn), and tau, which are detectable early in the preclinical phase and are already used diagnostically. CONTENT: The real-time quaking-induced conversion (RT-QuIC) assay is a thioflavin T-based in vitro method capable of detecting minute amounts of misfolded proteins in various biofluids with high sensitivity and specificity. It offers a promising approach for the early diagnosis of prion-like diseases. In recent years, RT-QuIC has been further developed, particularly to enhance the detection of αSyn-specific pathologies such as PD and related disorders. Additionally, increasing efforts have aimed to use peripheral and minimally invasive tissues or biofluids, such as skin, saliva, or nasal mucosa, for RT-QuIC analysis to support early and patient-friendly diagnostics. SUMMARY: This review summarizes recent advances in αSyn-specific RT-QuIC, with a particular focus on peripheral sample materials. The diagnostic potential of this technique for α-synucleinopathies and other NDDs is discussed and its future applications in biomarker discovery are explored.