Desipramine (DMI), a CYP2D6 probe, was used as a model drug to test whether CYP2D6-humanized (Tg-CYP2D6) and wild-type control mice could be used as preclinical animal models to identify the effects of CYP2D6 genotype/phenotype on drug metabolic profiles. After the analyses by liquid chromatography coupled with tandem mass spectrometry, DMI biotransformations were compared in Tg-CYP2D6 and wild-type mouse liver microsomes (MLM), and in human CYP2D6 extensive and poor metabolizer liver microsomes. Furthermore, urinary DMI metabolic profiles in Tg-CYP2D6 and wild-type mice were evaluated. Three metabolites, 2-hydroxyl-, 10-hydroxyl, and N-desmethyl-desipramine were identified in the incubations of DMI with both wild-type and Tg-CYP2D6 MLM, as well as in human CYP2D6 extensive metabolizer liver microsomes. Three additional metabolites were found in mouse urine samples, and their chemical structures were elucidated. Although the ratio of individual metabolites produced in Tg-CYP2D6 MLM was closer to that in human CYP2D6 extensive metabolizer liver microsomes, the urinary DMI metabolic profiles did not show much difference between wild-type and Tg-CYP2D6 mice. The results suggest that other mouse enzymes have significant contribution to DMI metabolism.