Alzheimer’s disease (AD) is the most common neurodegenerative disorder among older people, characterized by an insidious onset and a progressive decline of cognitive functions. Nowadays, there is no cure for AD mainly because its etiology is still unclear and current diagnostic tests show great limitations, including low sensitivity and specificity, as well as the impossibility to detect characteristic symptoms at early stages of disease. Thus, the main objective of this work was the optimization of complementary metabolomic approaches based on mass spectrometry in order to investigate AD pathogenesis and discover potential biomarkers for diagnosis. With the aim to get a comprehensive metabolome coverage, multiple analytical platforms were developed, including screening procedures based on direct mass spectrometry analysis and hyphenated approaches with orthogonal separation mechanisms such as liquid chromatography, gas chromatography and capillary electrophoresis. The application of these techniques to serum samples from patients suffering from Alzheimer’s disease and mild cognitive impairment enabled the identification of numerous metabolic alterations linked to pathogenesis of this disorder and its progression from pre-clinical stages, including abnormalities in the composition of membrane lipids, deficits in energy metabolism and neurotransmission, and oxidative stress, among others. Accordingly, it could be concluded that the combination of complementary metabolomic platforms allows studying etiology associated with Alzheimer’s disease in a deeper manner. See also slide presentation: https://sciforum.net/editor/submission/file/download/a92bcde043cd79bece22fe64e94831f8/slides