Background Real‐time quaking‐induced conversion (RT‐QuIC) and protein misfolding cyclic amplification (PMCA) have been developed to detect minute amounts of amyloidogenic proteins via amplification techniques and have been used to detect misfolded α‐synuclein (αSyn) aggregates in the cerebrospinal fluid (CSF) and other source materials of patients with Parkinson's Disease and other synucleinopathies. Objectives The aim of this systematic review and meta‐analysis was to evaluate the diagnostic accuracy of αSyn seed amplification assays (αSyn‐SAAs), including RT‐QuIC and PMCA, using CSF as source material to differentiate synucleinopathies from controls. Methods The electronic MEDLINE database PubMed was searched for relevant articles published until June 30, 2022. Study quality assessment was performed using the QUADAS‐2 toolbox. A random effects bivariate model was exploited for data synthesis. Results Our systematic review identified 27 eligible studies according to the predefined inclusion criteria, of which 22 were included in the final analysis. Overall, 1855 patients with synucleinopathies and 1378 non‐synucleinopathies as control subjects were included in the meta‐analysis. The pooled sensitivity and specificity to differentiate synucleinopathies from controls with αSyn‐SAA were 0.88 (95% CI, 0.82–0.93) and 0.95 (95% CI, 0.92–0.97), respectively. Evaluating the diagnostic performance of RT‐QuIC in a subgroup analysis for the detection of patients with multiple system atrophy the pooled sensitivity decreased to 0.30 (95% CI, 0.11–0.59). Conclusions While our study clearly demonstrated a high diagnostic performance of RT‐QuIC and PMCA for differentiating synucleinopathies with Lewy bodies from controls, results for the diagnosis of multiple system atrophy were less robust.