With advances in fast chromatography techniques, and highly sensitive and selective detection methods such as tandem mass spectrometry, very high-throughput bioanalytical methods can now be easily developed. The bottleneck of the analytical process then becomes the sample preparation, which it is now realized is crucial to the robust operation of the analytical system, especially for quantitative assays. Turbulent flow liquid chromatography was developed in the late 1990s, and combines ‘size exclusion’ and traditional stationary phase column chemistry to separate macromolecules, such as proteins, from smaller molecules and analytes of interest in biological fluids. By definition, the process is very rapid, and the instrumentation and software have been developed for fully automated, on-line extraction of neat biological fluids. This work aims to review the chromatographic theory of turbulent flow chromatography and illustrate, using examples from recent literature, the application of this technique to a range of analytes from a number of different biological matrices. Copyright © 2012 John Wiley & Sons, Ltd.