Deoxynivalenol (DON) is a mycotoxin that belongs to trichothecene subgroup B. Due its detrimental effects for human health, DON has been analyzed and detected by nearly all chromatographic methods. The present work reports for the first time the application of HPTLC/MS to confirm the presence of DON in wheat crops. Chromatography was performed on silica gel 60 F254 HPTLC plates using toluene - ethyl acetate - formic acid 6:3:1 (V/V/V) as mobile phase. After post-chromatographic derivatization with 10 % AlCl3, fluorescence detection was carried out at UV 366/>400 nm. Bands identity and purity was confirmed bymass spectrometry acquiring analytes directly from the sample bands by means of TLC/MS elution head-based interface. Both, sample and standard bands showed clear mass signals at m/z 341 that corresponds to the formate adduct of DON [M+HCOO]-. Thus, the presence of DON in the Chilean wheat sample was confirmed.