The analysis of bile salts in biological samples has remained a difficult task, due to the complex nature of the salts and also to their low concentration in common sample fluids such as plasma and urine. Given their importance, the development of accurate and sensitive methods of instrumental analysis has been the subject of intensive research, and recent advances have eliminated or lessened some of the difficulties. Currently available techniques are the following: thin-layer chromatography, gas chromatography, high-performance liquid chromatography, supercritical fluid chromatography, gas chromatography-mass spectrometry and capillary electrophoresis. Liquid chromatography coupled with mass spectrometry (thermospray, fast atom bombardment, electrospray and ionspray), a method undergoing continuous improvement, is also being applied to bile salts analysis. In this paper, these various techniques, which differ greatly in specificity, accuracy and simplicity, are reviewed and discussed, in terms of analytical performance, applicability to a given sample fluid, major limitations, ability to identify uncommon bile salts, including unsaturated oxo derivatives, glucuronides, sulfates, glycosides and bile alcohols.