The need to improve health outcomes in the developing world and to moderate healthcare costs in developed countries has resulted in an increased interest in sophisticated, inexpensive, and instrument-free point-of-care diagnostics using porous materials. One major segment of the paper-based diagnostics effort is focused on developing high-performance point-of-care tests using porous nitrocellulose membranes. This review provides a perspective on the nature, history, and future of nitrocellulose-based assays. Beginning as a protein blotting substrate, porous nitrocellulose membranes have grown to be the most commonly used lateral flow substrate and are the primary membranes used in two-dimensional paper networks for user-friendly multistep assays. In addition to the historical context, we examine assay development considerations, such as the physics of flow in porous media, reagent deposition and storage, and detection methods.