Due to the high daily intake of Annatto, a natural food colorant, it is important to determine its constituents in order to attain some information of nutritional significance. First, an Annatto extract was obtained. The ToF-SIMS spectra measured after depositing a drop of this extract on a silicon substrate exhibit strong molecular peaks clearly related to the bixin molecule (C HO), which has a molar mass M = 394. The most intense characteristic ion appears at m/z = 396 (M+2), but other molecular ions (e.g. at m/z = 337 obtained by the loss of COH or even a dimer at m/z = 790, or 2M + 2) were assigned to the bixin. On a silver substrate, M+Ag cations are also observed, confirming the straightforward identification of the colorant. The spectra recorded on the crude seed were remarkably similar to those obtained on the extract confirming the technique's ability to probe the seed's chemistry without preliminary treatment. After a prolonged storage in ambient light, a drastic reduction of the bixin molecular ion is observed, along with the appearance of xylene fragments.