The definitive diagnosis of human prion diseases can only be obtained postmortem by combining clinical symptoms, neuropathology and PrP immunohistochemistry, Western blotting of PrP types, zygosity of codon 129, and genetic study of PRNP. Premortem diagnosis is strongly sustained by one positive prion-specific assay, commonly protein misfolded cyclic amplification (PMCA) or real-time quaking-induced conversion (RT-QuIC), principally in CSF samples. Surrogate biomarkers 14-3-3, t-tau, P-tau, βA4, and total-PrP levels in the CSF help discriminate other neurodegenerative diseases, but their sensitivity and specificity are variable depending on the prion disease. Other altered proteins in the CSF, such as neurofilament light chain (NfL), calcium-binding protein S100β, neuron-specific enolase, α-synuclein and β-synuclein, neurogranin and SNAP-25, triggering receptor expressed on myeloid cells 2 (TREM2), cytokines, astroglial markers, and microRNAs, need further validation. Total-tau and NfL levels in the blood may serve to monitor disease progression, whereas the value of total-PrP, synuclein, S100β, TREM2, and peripheral inflammatory markers in the blood is limited. Since the products of positive PMCA and PrPSc are present in several tissues in CJD, special care and biosafety conditions must be applied in managing and processing human biological samples of suspected prion disease. Regarding RT-QuIC products, further experimental studies are needed to elucidate their seeding capacity.