Down's syndrome (DS) patients develop early Alzheimer's disease pathology with abundant cortical amyloid plaques, likely due to overproduction of the amyloid precursor protein (APP), which subsequently leads to amyloid β (Aβ) aggregation. This is reflected in cerebrospinal fluid (CSF) levels of the 42-amino acid long Aβ peptide (Aβ1-42), which are increased in young DS patients and decreases with age. However, it is unclear whether DS also affects other aspects of Aβ metabolism, including production of shorter C- and N-terminal truncated Aβ peptides, and production of peptides from the amyloid precursor-like protein 1 (APLP1), which is related to APP, and cleaved by the same enzymatic processing machinery. APLP1-derived peptides may be surrogate markers for Aβ1-42 production in the brain. Here, we used hybrid immunoaffinity-mass spectrometry and enzyme-linked immunosorbent assays to monitor several Aβ and APLP1 peptides in CSF from DS patients (n = 12) and healthy controls (n = 20). CSF levels of Aβ1-42 and three endogenous peptides derived from APLP1 (APL1β25, APL1β27 and APL1β28) were decreased in DS compared with controls, while a specific Aβ peptide, Aβ1-28, was increased in a majority of the DS individuals. This study indicates that DS causes previously unknown specific alterations of APP and APLP1 metabolism.