In vitro processing of neuropeptide Y (NPY) in cerebrospinal fluid (CSF) of patients with depression was monitored by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Single peptide bonds in NPY were cleaved to yield N- or C-terminal fragments. Multiple cleavage to form internal peptides was unimportant. Degradation rates varied between individuals, whereas the product distributions were fairly constant. Other peptides did not evidence such proteolysis. MALDI-TOF MS will facilitate extensive investigations of NPY processing that could provide the basis for clinical assays and illuminate the pathophysiology related to depression.