Abundant evidence suggests a central role for the amyloid-beta (Aβ) peptide in Alzheimer’s disease (AD) pathogenesis. Production and clearance of different Aβ isoforms have been established as targets of proposed disease-modifying therapeutic treatments of AD. However, previous studies used multiple sequential purification steps to isolate the isoforms individually and quantitate them based on a common mid-domain peptide. We created a method to simultaneously purify Aβ isoforms and quantitate them by the specific C-terminal peptides in order to investigate Aβ isoform physiology in the central nervous system. By using standards generated from in vitro metabolic labeling, the relative quantitation of four peptides representing total amount of Aβ (Aβ–Total), Aβ38, Aβ40, and Aβ42 were achieved both in cell culture and in human cerebrospinal fluid (CSF). Standard curves for each isoform demonstrated good sensitivity with very low limits of detection and high accuracy. Because the assay does not require antibody development for each Aβ isoform peptide, significant improvements in the throughput and accuracy of isoform quantitation were achieved.