The mycotoxin deoxynivalenol (DON) and some of its derivatives, such as 3‑acetyl‑deoxynivalenol (3AcDON), 15‑acetyl‑deoxynivalenol (15AcDON), deoxynivalenol‑3‑glucoside (DON3G) and de-epoxy deoxynivalenol (DOM-1), are commonly found in food and/or biological samples. However, literature does not present suitable methodologies for detecting and quantifying these mycotoxins at very low levels, which would be especially useful when they are present in biological samples. The main goal of the present paper was to evaluate different extraction techniques for the determination of these mycotoxins in rat faecal samples, in order to reduce the interferences present in the matrix and be able to quantify the mycotoxins at low concentration levels. Using diverse extraction methodologies such as QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) and pressurised liquid extraction (PLE), the clean-up strategy was optimised. QuEChERS extraction followed by a dispersive solid-phase extraction (dSPE) clean-up step with activated carbon was the method with the best extraction recovery results, ranging between 78% and 83% (except for DON3G). The matrix effect values were from -2% to -20% which supposed a reduction in comparison with the other tested strategies. These results enabled low quantification limits to be achieved, from 0.2 μg kg-1 to 3.4 μg kg-1. In view of the results, it was possible to quantify the natural presence of DON and DOM-1 in the tested faecal samples at low concentration levels.