Glucosinolates are phytochemicals with health promoting properties. Determination as desulpho-glucosinolates is widely used and adesulphation in microtiter plates has been applied to reach highthroughput. The use of various sulphatase concentrations and volumes throughout literature necessitates the identification of an appropriate desulphation procedure in microtiter plates. High sulphatase concentrations (¿15 mg/ml) decreased the concentration of the internal standard glucotropaeolin, whereas the other glucosinolates were less affected. Due to the calculation based on the recovery of glucotropaeolin, this leads to an overestimation of GL concentrations after desulphation with high sulphatase concentrations. A glucosidase side-activity, present in the crude sulphatase powder, is likely causing this phenomenon. At lower sulphatase concentrations (1 mg/ml) glucoiberin and glucoraphanin were insufficiently desulphated. Combining these effects results in a small range of applicable sulphatase concentrations. A purified sulphatase preparation resulted in good recoveries for a diversity of samples and is hence recommended for highthroughputdesulphation in microtiter plates.