Updated publication reference for PubMed record(s): 32132230. Laser-capture microdissection (LCM) allows the visualization and isolation of morphologically distinct subpopulations of cells from heterogeneous tissue specimens. In combination with formalin-fixed and paraffin-embedded (FFPE) tissue it provides a powerful tool for retrospective and clinically relevant studies of tissue proteins in a healthy and diseased context. In this study, we have developed an optimized protocol to facilitate efficient LCM analysis of FFPE tissue specimens. First, we optimized protein extraction from FFPE tissue by comparing different extraction buffers and investigating the influence of immunohistochemical and haematoxylin & eosin staining on proteins. SDS present in the protein extracts was removed with the SP3 digest method, which was modified to improve protein and peptide recoveries. Using a label-free approach protein expression of microdissected samples was compared to intact tissue sections from substantia nigra to evaluate the efficiency of LCM for the purification of small cell populations. The optimized protocol was used to analyse samples containing as few as ~3,000 cells isolated from the substantia nigra, using FFPE tissue. Replicate samples of 15 healthy donors were analysed in five separate TMT10plex batches, resulting in the quantification of >5,600 protein groups.