Isotopic internal standards are increasingly frequent in LC-MS analysis to control biological matrix effects in the quantitation of immunosuppressant drugs, such as everolimus (RAD001). Here we present the evaluation of a LC-MS method, exploiting [13C2D4]RAD001 as internal standard, for preclinical determination of RAD001 in mice brain tissue. Samples were purified by solid phase extraction. Brain and blood were collected from vehicle-treated and RAD001-treated mice. The QTOF MS detector was set to select RAD001 ammonium adducts (m/z 975.6152) and [13C2D4]RAD001 (m/z 981.6481). Two different UHPLC columns were preliminarily tested. The method showed linear behavior between 4-100 ng/mL (r2 = 0.99943) and linearity was preserved in the presence of blood (r2 = 0.99107) and brain (r2 = 0.99098) matrix components. Intra-day and inter-day precision (3%-19%) and accuracy (82%-109%) were comparable between standards and spiked blood and brain samples. As resulting from recovery comparison (82%-98%), [13C2D4]RAD001 compensated ion suppression phenomena maintaining method performance over a wide range of consecutive analytical runs. The comparison with a HPLC-UV method showed reliability of the method with good correlation between blood (r2 = 0.94319) and brain (r2 = 0.97773) samples and acceptable biases (<15%). This validation suggests that the investigated method could be useful for the preclinical monitoring of RAD001 brain therapeutic concentrations in animal models.