Aim: There is an unmet need for compounds that detect alpha-synuclein (αSyn) and 4-repeat tau, which are critical in many neurodegenerative diseases for diagnostic and therapeutic purposes. Here, we aim to develop an efficient surface plasmon resonance (SPR)-based method to facilitate the characterization of small molecule ligands/compounds to these fibrils. Methods: SPR measurements were conducted to characterize the binding properties of fluorescent ligands/compounds towards recombinant Aβ42, K18 4-repeat/full-length tau and αSyn fibrils. In silico modelling was performed to examine the binding pockets of ligands on αSyn fibrils. Immunofluorescence staining with fluorescence ligands and specific antibodies on postmortem brain tissue slices from patients with Parkinson′s disease and disease mouse models was performed. Results: We optimized the protocol for immobilizing Aβ42, K18 tau, full-length tau and αSyn fibrils in a controlled aggregation state on SPR sensor chips. The results from the analysis of binding kinetics suggested the presence of at least two binding sites for all fibrils, including luminescent conjugated oligothiophenes (HS-169, HS-84, h-FTAA and q-FTAA), pyridine derivative PBB5, nonfluorescent methylene blue and lansoprazole. In silico modelling studies for αSyn (6H6B) showed four binding sites with preference to S4. Immunofluorescence staining validated the detection of pS129-positive αSyn in brain tissue from Parkinson′s disease patients, αSyn PFF-injected mice, 6E10-positive Aβ in arcAβ mice, and AT-8/AT-100-positive in tau pR5 tau mice, respectively. Conclusions: SPR measurements of ligands and small molecules binding to Aβ42, 4R- and full-length tau and αSyn fibrils suggest the existence of multiple binding sites. This approach may provide efficient characterization of compound binding properties towards these fibrils important in neurodegenerative diseases.