Synucleinopathies are a group of clinically and neuropathologically heterogenous disorders characterized by the abnormal aggregation of α-synuclein in the form of Lewy bodies, Lewy neurites, and glial cytoplasmic inclusions. The ante-mortem diagnosis of synucleinopathies relies mostly on clinical features. However, clinical manifestations appear several years after the onset of neuropathological changes, with a considerable degree of overlap in clinical phenotypes across these disorders. Biomarkers reflecting α-synuclein pathology could allow for accurate and early diagnosis, also serving prognostic purposes, i.e., identifying those patients with the highest risk of clinical progression. With respect to clinical trials with disease-modifying treatments, biomarkers reflecting α-synuclein pathology could be valuable to include biologically homogeneous patients in which the molecular target of the therapy is actually represented, also providing outcome measures to verify the biological impact of treatment. In this chapter, current evidence on fluid and imaging biomarkers reflecting α-synuclein pathology will be discussed. Among fluid biomarkers, seeding amplification assays, which are able to identify α-synuclein prone to aggregation in cerebrospinal fluid and other peripheral tissues, so far, appear to offer the most promising techniques for in vivo identification of synucleinopathies. Clinical application of α-synuclein imaging of the brain is limited by several technical issues. To date, the combination of different biological matrices and the association of α-synuclein with other biomarkers could pave the way for an early and accurate diagnosis of synucleinopathies and better prognostication of associated clinical outcomes.