There is an increasing need of new bio-analytical methodologies with enough sensitivity, robustness and resolution to cope with the analysis of a large number of analytes in complex matrices in short analysis time. For this purpose, all steps included in any bio-analytical method (sampling, extraction, clean-up, chromatographic analysis and detection) must be taken into account to achieve good and reliable results with cost-effective methodologies. The purpose of this review is to describe the state-of-the-art of the most employed technologies in the period 2009-2012 to achieve fast analysis with liquid chromatography coupled to mass spectrometry (LC-MS) methodologies for bio-analytical applications. Current trends in fast liquid chromatography involve the use of several column technologies and this review will focus on the two most frequently applied: sub-2 µm particle size packed columns to achieve ultra high pressure liquid chromatography (UHPLC) separations and porous-shell particle packed columns to attain high efficiency separations with reduced column back-pressures. Additionally, recent automated sample extraction and clean-up methodologies to reduce sample manipulation, variability and total analysis time in bio-analytical applications such as on-line solid phase extraction coupled to HPLC or UHPLC methods, or the use of other approaches such as molecularly imprinted polymers, restricted access materials, and turbulent flow chromatography will also be addressed. The use of mass spectrometry and high or even ultra-high resolution mass spectrometry to reduce sample manipulation and to solve ion suppression or ion enhancement and matrix effects will also be presented. The advantages and drawbacks of all these methodologies for fast and sensitive analysis of biological samples are going to be discussed by means of relevant applications.