Cell migration requires the formation of a leading pseudopodium (lamellipodium) in the direction of movement. This process requires signal amplification to facilitate directional sensing mechanisms that lead to actin-mediated membrane extension. However, it has been difficult to study pseudopodia formation because it has not been possible to purify this structure for biochemical analysis. Here we describe a method to biochemically purify the protruding pseudopodium from the cell body compartment using polycarbonate microporous filters. Cells are cultured on top of 3.0-microm porous filters and allowed to extend pseudopodia through the small pores to the undersurface in response to a gradient of either chemokine or extracellular matrix (ECM) protein. Pseudopodia and cell bodies are then differentially scraped from the filter surface into lysis buffer for biochemical analysis. Using this method, it is possible to identify novel pseudopodium and cell body proteins as well as study the spatiotemporal organization of signaling processes that regulate pseudopodium formation and cell polarity. This method will help facilitate our understanding of how cells protrude pseudopodia through small openings in the ECM and vasculature during cancer cell invasion, immune cell surveillance, and embryonic development.