Miniaturized separation techniques have emerged as environmentally friendly alternatives to available separation methods. Nano-liquid chromatography (nano-LC), microchip devices and nano-capillary electrophoresis are miniaturized methods that minimize reagent consumption and waste generation. Furthermore, the low levels of analytes, especially in biological samples, promote the search for more highly sensitive techniques; coupled to mass spectrometry, nano-LC has great potential to become an indispensable tool for routine analysis of biomolecules. This short review presents the fundamental aspects of nano-LC analytical instrumentation, discussing practical considerations and the primary differences between miniaturized and conventional instrumentation. Some theoretical aspects are discussed to better explain both the potential and the principal limitations of nano-LC. Recent pharmaceutical and biomedical applications of this separation technique are also presented to indicate the satisfactory performance for complex matrices, especially for proteomic analysis, that is obtained with nano-LC.