Focal adhesions (FAs) are discrete plasma membrane-associated adhesive organelles that play dual roles in cell force transduction and signaling. FAs consist of clustered transmembrane heterodimeric integrin extracellular matrix (ECM) receptors and a large number of cytoplasmic proteins that collectively form thin plaques linking the ECM to actin filament bundles of the cytoskeleton. FAs are complex organelles that can change their composition in response to biochemical or mechanical cues. These compositional differences may underlie the ability of FAs to mediate an array of important cell functions including adhesion, signaling, force transduction, and regulation of the cytoskeleton. These functions contribute to the physiological processes of the immune response, development, and differentiation. However, linking FA composition to FA function has been difficult since there has been no method to isolate intact FAs reproducibly and determine their composition. We report here a new method for isolating FA structures in cultured cells distinct from cytoplasmic, nuclear, and internal membranous organellar components of the cell. We provide protocols for validation of the fractionation by immunofluorescence and immunoblotting, procedures for preparing the isolated FAs for mass spectrometric proteomic analysis, tips on data interpretation and analysis, and an approach for comparing FA composition in cells in which small GTPase signaling is perturbed.