Amyloid-β (Aβ) producing enzymes are key targets for disease-modifying Alzheimer's disease (AD) therapies since Aβ trafficking is at the core of AD pathogenesis. Development of such drugs might benefit from the identification of markers indicating in vivo drug effects in the central nervous system. We have previously shown that Aβ(1-15) is produced by concerted β-and α-secretase cleavage of amyloid-β protein precursor (AβPP). Here, we test the hypothesis that this pathway is more engaged upon γ-secretase inhibition in humans, and cerebrospinal fluid (CSF) levels of Aβ(1-15/16) represent a biomarker for this effect. Twenty healthy men were treated with placebo (n = 5) or the γ-secretase inhibitor semagacestat (100 mg [n = 5], 140 mg [n = 5], or 280 mg [n = 5]). CSF samples were collected hourly over 36 hours and 10 time points were analyzed by immunoassay for Aβ(1-15/16), Aβ(x-38), Aβ(x-40), Aβ(x-42), sAβPPα, and sAβPPβ. The CSF concentration of Aβ(1-15/16) showed a dose-dependent response over 36 hours. In the 280 mg treatment group, a transient increase was seen with a maximum of 180% relative to baseline at 9 hours post administration of semagacestat. The concentrations of Aβ(x-38), Aβ(x-40), and Aβ(x-42) decreased the first 9 hours followed by increased concentrations after 36 hours relative to baseline. No significant changes were detected for CSF sAβPPα and sAβPPβ. Our data shows that CSF levels of Aβ(1-15/16) increase during treatment with semagacestat supporting its feasibility as a pharmacodynamic biomarker for drug candidates aimed at inhibiting γ-secretase-mediated AβPP-processing.