The seasonal fluctuation of glucosinolates in five defined Isatis tinctoria and one Isatis indigotica accessions (first year, rosette stage), grown on field plots under identical conditions, was investigated. Analysis of the intact glucosinolates was carried out with shock frozen, freeze dried leaf samples using a recently developed and validated PLE (pressurized liquid extraction) protocol and ion-pair HPLC coupled with ESI-MS in the negative mode. When comparing the two Isatis species, significant qualitative and quantitative differences in the glucosinolate patterns were observed. Differences among the various Isatis tinctoria accessions were much smaller. We studied the effects of repeated harvesting during the growth season on glucosinolate concentrations and found that repeated harvest did not have a major effect on glucosinolate concentrations of newly grown leaves. Glucosinolates could not be detected in woad leaves submitted to conventional drying.