Amyloid-Beta (Aβ) peptides in human Plasma containing Aβ42 (residues 1–42) and Aβ40 (residues1–40), are utilized as biomarkers in work for the diagnosis of Alzheimer’s disease. For application to drug (Chemical drug, Biopharmaceutical etc.) efficacy test using animal model (2XFAD / 5XFAD mice model), Aβ42 (residues 1–42) and Aβ40 (residues1–40) was determined in mouse plasma by LC-MS/MS using isotopic as an internal standard. To simplification of extraction steps and optimization of chromatographic condition, sample preparation method was accomplished using solid phase extraction. The LC-MS/MS system was equipped with an electrospray source operating in the positive ion mode. The lower limit of quantification (LLOQ) was 30 pg/mL, using a sample volume of 100 μL for the analysis. The reproducibility of the method was evaluated by analyzing five replicates at four quality control (QC) levels over the nominal concentration range Aβ42 and Aβ40, 30 to 400 pg/mL.