The extracellular matrix acts as a framework for tissue architecture and dynamically regulates many cellular functions. Fibronectin is a ubiquitous extracellular matrix component that plays critical roles in matrix structure and in directing cell behaviors. Fibronectin is synthesized and secreted by many cell types including fibroblasts, endothelial cells, myoblasts, and astrocytes. Upon secretion, cells assemble fibronectin into a fibrillar network. During assembly, fibronectin is initially organized into fine cell-associated fibrils and, through continued accumulation of fibronectin, these fibrils are converted into a dense network of detergent-insoluble fibrils. Differential solubility in the detergent deoxycholate is the principle for biochemical fractionation of fibronectin matrix. Fibril assembly and organization can also be examined by immunofluorescence staining. In this unit, basic methods of detection, quantification, and visualization of fibrillar fibronectin matrix are described.