A new, rapid and sensitive method has been developed for the determination of nivalenol (NIV) and deoxynivalenol (DON) by using HPLC in combination with an atmospheric pressure chemical ionization (APCI)-interface and a single quadrupole mass spectrometer. Different LC and MS parameters have been optimized prior to this in order to obtain better results and sensitivity. The effect of nebulizing temperature on the sensitivity and fragmentation of NIV and DON in an APCI interface was investigated. Also, the influence of the cone voltage on the fragmentation pattern was studied, which was shown to have a tremendous effect. Furthermore, the effect of modifiers such as ammonium acetate, acetic acid and ammonia on the ionisation yield of the above substances have been investigated. The extraction was carried out using acetonitrile-water. A two step purification was then applied on two different Mycosep clean up columns. We have used a modified, rapid and isocratic HPLC method combined with a negative ion APCI-MS for the separation and quantitative determination of NIV and DON in wheat extract. An RP C18 column was used for the separation of selected compounds in wheat extract with water-acetonitrile-methanol (82:9:9, v/v/v) at a flow-rate of 1 ml/min without a split. Calibration curves show good linearity and reproducibility. The detection limit and precision were determined for NIV and DON. Both compounds could be detected down to microg/kg level in wheat using selected ion monitoring of the [M-H]- ions and the main fragments.