The purpose of this study was to identify the protein profile of the mouse brain microvascular endothelium in situ. This involved coupling of a double-label, immuno-laser capture microdissection (LCM) process with LTQ-FT mass spectrometry to perform the in situ proteomic analysis. LCM was utilized to isolate cells from frozen mouse brain tissue sections. Following cell capture, samples were solubilized and proteins separated by gel electrophoresis in preparation for enzymatic digestion and LC-MS analysis. Processed samples were subsequently analyzed using a linear IT coupled with a Fourier transform mass spectrometer (LTQ-FT MS). Overall, in this study, 881 proteins were identified from a specific cell category using immuno-guided LCM to probe these cell types along the entirety of the cerebral microvascular tree. The identification of sufficient numbers of proteins with high biological interest should allow us to study protein expression by specific cell types - as defined by certain cell markers - in complex tissues.