Alzheimer's disease (AD) currently affects more than 30 million people worldwide. The lack of understanding of AD's physiopathology limits the development of therapeutic and diagnostic tools. Soluble amyloid-β peptide (Aβ) oligomers that appear as intermediates along the Aβ aggregation into plaques are considered among the main AD neurotoxic species. Although a wealth of data are available about Aβ from in vitro and animal models, there is little known about intracellular Aβ in human brain cells, mainly due to the lack of technology to assess the intracellular protein content. The elucidation of the Aβ species in specific brain cell subpopulations can provide insight into the role of Aβ in AD and the neurotoxic mechanism involved. Here, we report a microfluidic immunoassay for in situ mass spectrometry analysis of intracellular Aβ species from archived human brain tissue. This approach comprises the selective laser dissection of individual pyramidal cell bodies from tissues, their transfer to the microfluidic platform for sample processing on-chip, and mass spectrometric characterization. As a proof-of-principle, we demonstrate the detection of intracellular Aβ species from as few as 20 human brain cells.