Mass spectrometry imaging (MSI) is well-known for the non-labeling visualization ofanalytes, including drugs and their metabolites in biological samples. In this study, we appliedthree different tools of MSI, desorption electrospray ionization (DESI)-MSI, matrix-assisted laserdesorption ionization (MALDI)-MSI, and a newly developed atmospheric pressure (AP)-MALDI-MSIknown as iMScopeTM QT for rapid mapping of imipramine, chloroquine, and their metabolites inC57BL/6 male wild-type mice. Among three MSI tools, better detection capability for targeted drugsat higher speed (up to 32 pixels/s) was observed in iMScope QT. It revealed that imipramine andits metabolites were significantly accumulated in the renal cortex of mice, but chloroquine and itsmetabolites were highly accumulated in the renal pelvis and renal medulla of mice. Additionally, ahigher accumulation of imipramine was noted in the thalamus, hypothalamus, septum, and hindbrainof mice brains. However, chloroquine and its metabolites showed notable accumulation in the lateralventricle, fourth ventricle, and fornix of the mice brains. These findings of our study can be helpfulin understanding clinically relevant properties, efficacy, and potential side effects of these drugs.Our study also showed the potentiality of iMScope QT for rapid mapping of small drugs and theirmetabolites in biological samples.