Amyloidogenic peptides such as the Alzheimer’s disease-implicated Amyloid beta (Aβ), can present a significant challenge when trying to obtain high purity material. Here we present a tailored HPLC purification protocol to produce high-purity amyloid beta 42 (Aβ42) and amyloid beta 40 (Aβ40) peptides. We have found that the combination of commercially available hydrophobic poly(styrene/divinylbenzene) stationary phase, polymer laboratory reverse phase - styrenedivinylbenzene (PLRP-S) under high pH conditions, enables the attainment of high purity (>95%) Aβ42 in a single chromatographic run. The purification is highly reproducible and can be amended to both semi-preparative and analytical conditions depending upon the amount of material wished to be purified. The protocol can also be applied to the Aβ40 peptide with identical success and without the need to alter the method. © 2017 Journal of Visualized Experiments.