Amphiphile self-assembly materials, which contain both a hydrophilic and a hydrophobic domain, have great potential in high-throughput and combinatorial approaches to discovery and development. However, the materials chemistry community has not embraced these ideas to anywhere near the extent that the medicinal chemistry community has. While this situation is beginning to change, extracting the full potential of high-throughput approaches in the development of self-assembling materials will require further development in the synthesis, characterization, formulation, and application domains. One of the key factors that make small molecule amphiphiles prospective building blocks for next generation multifunctional materials is their ability to self-assemble into complex nanostructures through low-energy transformations. Scientists can potentially tune, control, and functionalize these structures, but only after establishing their inherent properties. Because both robotic materials handling and customized rapid characterization equipment are increasingly available, high-throughput solutions are now attainable. These address traditional development bottlenecks associated with self-assembling amphiphile materials, such as their structural characterization and the assessment of end-use functional performance. A high-throughput methodology can help streamline materials development workflows, in accord with existing high-throughput discovery pipelines such as those used by the pharmaceutical industry in drug discovery. Chemists have identified several areas that are amenable to a high-throughput approach for amphiphile self-assembly materials development. These allow an exploration of not only a large potential chemical, compositional, and structural space, but also material properties, formulation, and application variables. These areas of development include materials synthesis and preparation, formulation, characterization, and screening performance for the desired end application. High-throughput data analysis is crucial at all stages to keep pace with data collection. In this Account, we describe high-throughput advances in the field of amphiphile self-assembly, focusing on nanostructured lyotropic liquid crystalline materials, which form when amphiphiles are added to a polar solvent. We outline recent progress in the automated preparation of amphiphile molecules and their nanostructured self-assembly systems both in the bulk phase and in dispersed colloidal particulate systems. Once prepared, we can structurally characterize these systems by establishing phase behavior in a high-throughput manner with both laboratory (infrared and light polarization microscopy) and synchrotron facilities (small-angle X-ray scattering). Additionally, we provide three case studies to demonstrate how chemists can use high-throughput approaches to evaluate the functional performance of amphiphile self-assembly materials. The high-throughput methodology for the set-up and characterization of large matrix in meso membrane protein crystallization trials can illustrate an application of bulk phase self-assembling amphiphiles. For dispersed colloidal systems, two nanomedicine examples highlight advances in high-throughput preparation, characterization, and evaluation: drug delivery and magnetic resonance imaging agents.