Amyloid-β (Aβ) peptides, including post-translationally modified variants thereof, are believed to play a key role in the onset and progression of Alzheimer's disease. Suggested modified Aβ species with potential disease relevance include Aβ peptides phosphorylated at serine in position eight (pSer8-Aβ) or 26 (pSer26-Aβ). However, the published studies on those Aβ peptides essentially relied on antibody-based approaches. Thus, complementary analyses by mass spectrometry, as shown for other modified Aβ variants, will be necessary not only to unambiguously verify the existence of phosphorylated Aβ species in brain samples but also to reveal their exact identity as to phosphorylation sites and potential terminal truncations. With the aim of providing a novel tool for addressing this still-unresolved issue, we developed a customized matrix formulation, referred to as TOPAC, that allows for improved detection of synthetic phosphorylated Aβ species by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. When TOPAC was compared with standard matrices, we observed higher signal intensities but minimal methionine oxidation and phosphate loss for intact pSer8-Aβ(1-40) and pSer26-Aβ(1-40). Similarly, TOPAC also improved the mass spectrometric detection and sequencing of the proteolytic cleavage products pSer8-Aβ(1-16) and pSer26-Aβ(17-28). We expect that TOPAC will facilitate future efforts to detect and characterize endogenous phosphorylated Aβ species in biological samples and that it may also find its use in phospho-proteomic approaches apart from applications in the Aβ field.