〔Abstract〕 The mass-spectrometry-based binding assays(MS binding assays,MSBA) would be established to determine the affinity of small-molecule probes with amyloid-β(Aβ) aggregates in this study.The liquid chromatography-mass spectrometry(LC-MS)-based method and quantitative method were established for the existing positive probe(IMPY) and its deuterated product(IMPY-D6),followed by mass spectrometry-based saturation binding experiments and competitive binding experiments.Finally,the experimental data were analyzed and compared with the results of the radioligand binding assays.The LC-MS method of IMPY and MSBA were established successfully.In the saturation binding experiments,the Kdfor IMPY with Aβ 1-40 and Aβ 1-42 were(3.80±0.39) nmol/L and(18.38±1.11) nmol/L,respectively.In the competitive binding experiments,the Kifor IMPY with Aβ 1-40 and Aβ 1-42 were(18.64±0.76) nmol/L and(25.44±2.38) nmol/L,respectively.These data were consistent with the results of the radioligand binding assays.As a non-radioactive binding experiment method,the results of MSBA were consistent with the radioligand binding assays and could be used for the affinity detection of small-molecule probes to Aβ.This technique can be extended to the determination experiment of the affinity between conventional small-molecule ligands and receptors.