Formalin-fixed paraffin-embedded (FFPE) tissue, while excellent for preserving tissue for extended periods of time, poses a challenge when extracting molecular information. We therefore developed an easily adaptable and highly efficient workflow for extracting high levels of proteins from low-input material. We compared sensitivity between two stains, EpCAM and H&E, across material inputs of ~2 and 1,600 cells. In the context of EpCAM-stained tissue, our investigations unveiled a range from ~1,200 unique protein groups at the lowest input to ~5,900 at the highest. For H&E, the spectrum covers ~900 to ~5,200 protein groups. We found an optimal balance between maximizing detected proteins and minimizing input material to be within the range of ~100 to ~200 cells. With this knowledge, we tested the spatial capabilities by isolating specific cell populations, through Laser Capture Microdissection (LCM), from three different tissue types, where we were able to identify tissue-specific signatures and prominent clustering of all cell populations.