This review article discusses the impact of nanostructured stationary phases on liquid chromatography and separation science. These materials have revolutionized chromatography by enabling unprecedented levels of sensitivity, resolution, and applicability. Nanoporous silica, graphenic, monolithic, and nanoparticle-based phases continue to push the boundaries of biomolecular analysis, molecular diagnostics, and traceability testing. Nanostructured phases have made early detection of diseases, comprehensive profiling of proteomes, enhanced food origin traceability, and sensitive environmental monitoring possible. They facilitate isolation and analysis of biomacromolecules, extracellular vesicles, viruses, and trace constituents with high specificity and sensitivity even from minimal sample volumes. Furthermore, nanostructured phases are enabling integrated techniques, sensing capabilities, and responsive microdomains for advanced detection, purification, and separation of analytes. Continued progress in nanomaterial design, surface engineering, and micro-nanofabrication will lead to more sophisticated nano-LC approaches with translation across healthcare, food safety, materials analysis, and global sustainability. The review concludes that nanostructured stationary phases represent a pivotal frontier in chromatography and analytical sciences with tremendous potential to transform molecular diagnosis, precision medicine, origin traceability, and monitoring of health, food, and environment quality. Nano-LC promises to make comprehensive and minimally invasive molecular-level understanding more feasible, accessible, and impactful. These materials are an enabling technology with immense and far-reaching possibilities that will likely shape developments in analytical sciences and their use for years to come.