An important pathological hallmark of Alzheimer's disease (AD) is the deposition of amyloid-beta (A) peptides in the brain parenchyma, leading to neuronal death and impaired learning and memory. The protease -secretase is responsible for the intramembrane proteolysis of the amyloid- precursor protein (APP), which leads to the production of the toxic A peptides. Thus, an attractive therapeutic strategy to treat AD is the modulation of the -secretase activity, to reduce A42 production. Because phosphorylation of proteins is a post-translational modification known to modulate the activity of many different enzymes, we used electrospray (LC-MS/MS) mass spectrometry to identify new phosphosites on highly purified human -secretase. We identified 11 new single or double phosphosites in two well-defined domains of Presenilin-1 (PS1), the catalytic subunit of the -secretase complex. Next, mutagenesis and biochemical approaches were used to investigate the role of each phosphosite in the maturation and activity of -secretase. Together, our results suggest that the newly identified phosphorylation sites in PS1 do not modulate -secretase activity and the production of the Alzheimer's A peptides. Individual PS1 phosphosites shall probably not be considered therapeutic targets for reducing cerebral A plaque formation in AD.