Prion diseases, a group of fatal neurodegenerative disorders, present significant diagnostic challenges due to their subtle clinical manifestations, which often mimic those of other treatable neurological conditions. This diagnostic ambiguity underscores the necessity for reliable and specific diagnostic tools. Real-Time Quaking-Induced Conversion (RT-QuIC) has emerged as a groundbreaking technique for diagnosing these diseases using cerebrospinal fluid (CSF) samples. This chapter outlines a comprehensive and standardized protocol for employing RT-QuIC to detect the pathological form of the prion protein in CSF, offering a powerful tool for clinical diagnosis. It provides step-by-step instructions for patient examinations, laboratory RT-QuIC analysis, and data interpretation. By adhering to these protocols, researchers and clinicians can achieve high sensitivity and specificity in diagnosing prion diseases, facilitating timely and accurate patient management.