AbstractLow‐flow chromatography has a rich history of innovation but has yet to reach widespread implementation in bioanalytical applications. Improvements in pump technology, microfluidic connections, and nano‐electrospray sources for MS have laid the groundwork for broader application, and innovation in this space has accelerated in recent years. This article reviews the instrumentation used for nano‐flow LC, the types of columns employed, and strategies for multidimensionality of separations, which are key to the future state of the technique to the high‐throughput needs of modern bioanalysis. An update of the current applications where nano‐LC is widely used, such as proteomics and metabolomics, is discussed. But the trend toward biopharmaceutical development of increasingly complex, targeted, and potent therapeutics for the safe treatment of disease drives the need for ultimate selectivity and sensitivity of our analytical platforms for targeted quantitation in a regulated space. The selectivity needs are best addressed by mass spectrometric detection, especially at high resolutions, and exquisite sensitivity is provided by nano‐electrospray ionization as the technology continues to evolve into an accessible, robust, and easy‐to‐use platform.