Despite the increasing success of liquid chromatography (LC) coupled to mass spectrometry (MS), matrix effects have limited the ESI applications. Matrix effects result from co-eluting residual matrix components affecting the ionization efficiency of target analytes and can lead to erroneous results. The present work evaluates the LC-electrospray ionization (ESI)-MS behaviour of benzoxazinoid derivatives in wheat plant material as a case where endogenous matrix components were found to interfere with the ionization of target analytes. When LC-MS was applied to the analysis of foliage and root extracts, significant signal suppressions were observed. To compensate for this matrix signal suppression, different methods were applied: the use of internal standards, the application of standard dilution method and the dilution of the extracts before instrumental determination. The results obtained indicated that evaluation of matrix effects should become an integrated part of quantitative LC-ESI-MS method development and validation.