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The ratio of amyloid precursor protein (APP)_669–711 (Aβ_−3–40)/Aβ_1–42 in blood plasma was reported to represent a novel Alzheimer’s disease biomarker. Here, we describe the characterization of two antibodies against the N-terminus of Aβ_−3–x and the development and “fit-for-purpose” technical validation of a sandwich immunoassay for the measurement of Aβ_−3–40. Antibody selectivity was assessed by capillary isoelectric focusing immunoassay, Western blot analysis, and immunohistochemistry. The analytical validation addressed assay range, repeatability, specificity, between-run variability, impact of pre-analytical sample handling procedures, assay interference, and analytical spike recoveries. Blood plasma was analyzed after Aβ immunoprecipitation by a two-step immunoassay procedure. Both monoclonal antibodies detected Aβ_−3–40 with no appreciable cross reactivity with Aβ_1–40 or N-terminally truncated Aβ variants. However, the amyloid precursor protein was also recognized. The immunoassay showed high selectivity for Aβ_−3–40 with a quantitative assay range of 22 pg/mL–7.5 ng/mL. Acceptable intermediate imprecision of the complete two-step immunoassay was reached after normalization. In a small clinical sample, the measured Aβ₄₂/Aβ_−3–40 and Aβ₄₂/Aβ₄₀ ratios were lower in patients with dementia of the Alzheimer’s type than in other dementias. In summary, the methodological groundwork for further optimization and future studies addressing the Aβ₄₂/Aβ_−3–40 ratio as a novel biomarker candidate for Alzheimer’s disease has been set.