Amongst a variety of non-digestible oligosaccharides, galactooligosaccharides (GOS) prebiotic condition is supported by strong scientific evidence and they have been definitely established as prebiotics. They can beneficially affect the host by stimulating growth of selected members of the intestinal microflora, reducing, thus, the formation of toxic metabolites, and producing short chain fatty acids. GOS are principally synthesized by enzymatic transgalactosylation reaction from lactose by various microorganisms (e.g.: Kluyveromyces genus or Pseudozyma tsukubaensis), obtaining complex mixtures of oligosaccharides with different glycosidic linkages and degree of polymerization. The structure and product ratio of GOS obtained by transgalactosylation reactions depend on the biocatalyst as well as on the conditions employed. The aim of this study was to identify GOS produced from P. tsukubaensis by MALDI-TOFMS. Erlenmeyer flasks containing 100mL of a medium composed by 40% lactose (w/v), 0.5% yeast extract (w/v) and 0.1% (w/v) urea were inoculated with 0.02155g of cells per 100 mL (7% v.v-1).The flasks were placed in a rotary shaker (30oC/150rpm) for 36h and then centrifuged 104g. The supernatant was analyzed using MALDI-TOFMS. 1μL of samples was placed on the target plate, then 1μL of matrix solution (saturated α-hydroxycinnaminic acid in 0.1% TFA:acetonitrile 1:1) was placed on the samples. MALDI-TOFMS spectra were recorded by using a smartbeanTM laser for desorption and ionization. Matrix-suppression was also used, and the mass spectra were averaged over 50 to 100 individual laser shots. GOS were anticipated to have an m/z range of 300–900. The MALDI-TOF mass spectrometry data revealed that at least two GOS molecules (≈527m/z trisaccharides and ≈689m/z tetrasaccharides) were present, in which the tetrasaccharides were a smaller peak. It was also observed a presence of adducts of lactose (≈360m/z). To the best of our knowledge, this is the first study reporting the identification of GOS produced from P. tsukubaensis by MALDI-TOFMS.