The determination of glucosinolates by high-performance liquid chromatography (HPLC) is well established in many laboratories, and efforts have been made to work out a suitable HPLC standard method to replace the current tedious gas-liquid chromatographic (GLC) method. The HPLC methods are especially well suited for indolyl glucosinolates which can easily be missed in GLC analyses. Introduction of the new 00-varieties of rapeseed containing low amounts of glucosinolates has made it essential to measure not only the alkenylglucosinolates but also the more complex, i.e., indolylglucosinolates. Most HPLC separations of glucosinolates are done after an enzymatic desulphation step producing non-ionic desulphoglucosinolates, which are well suited for reversed-phase (RP) HPLC. Desulphation is performed on an ion-exchange column that also serves for the sample clean-up step, but complete desulphation takes at least 12 h; desulphation in solution takes 20 min but no sample clean-up. Special attention has to be paid to temperature, pH, water amount, and sulphatase quality in desulphation. An early method for separating intact glucosinolates by ion-pair HPLC avoiding desulphation has been described and improved. Most laboratories use water-acetonitrile gradient for desulphoglucosinolates with UV detection, and other eluents like water-methanol or acetone-ethyl acetate-water have been used. Adding neutral salts to the eluent in reversed-phase chromatography increases retention of anionic species. This phenomenon is utilized in separating intact glucosinolates using aqueous ammonium acetate-acetonitrile mixtures instead of water-acetonitrile, allowing separation and determination without tedious desulphation and expensive ionpair reagents.