Primary aldosteronism (PA) is the main cause of secondary hypertension, accounting for approximately 5–10% of all hypertension. Amlodipine, a third-generation calcium channel blocker, is one of the most frequently administered pharmaceuticals medications of hypertension, binds specifically to Cav1.2, a calcium channel primarily localized in the cardiovascular system, and exerts antihypertensive effects through inhibiting calcium influx into the vascular smooth muscle cells. In addition, calcium influx also plays important roles in aldosterone production and amlodipine was also reported to influence in vitro functions of Cav1.3, a calcium channel involved in aldosterone secretion. Ca channel blockers were also reported to reduce plasma aldosterone concentration by some clinical studies although with mild degrees. However, in vivo effects of amlodipine to aldosterone secretion has remained virtually unknown. A novel technique “Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging (MALDI-MSI)” has been recently developed, which did make it possible to visualize non-labeled small molecules on tissue sections. Therefore, in this study, we firstly applied MALDI-MSI to visualize amlodipine on human adrenal glands including aldosterone producing adenoma (APA). We performed selective imaging of amlodipine using MALDI-MSI on the resected adrenal tissues from APA patients. Frozen sections containing whole representative tumor area were coated with a matrix called CHCA (α-Cyano-4-hydroxycinnamic acid) by deposition as a pretreatment. We subsequently analyzed and detected a precursor ion with MS at m/z 407.1 and then an amlodipine-specific ion with MS/MS at m/z 318.1. We also examined the concordance of amlodipine distribution obtained by this method with immunohistochemistry. Human resected adrenal tissues obtained from the patients APAs treated with and without amlodipine before adrenalectomy were examined. Periadrenal adipose tissues were also analyzed as a control tissue of non-aldosterone-producing tissues. Amlodipine was specifically detected and visualized only in the administered cases. Amlodipine was more abundantly detected in adrenal tissues than periadrenal adipose tissues. On the other hand, significant different was not detected between tumors and adjacent adrenal glands by semi-quantification using MALDI-MSI. In this study, we firstly visualized amlodipine directly in human tissue sections using MALDI-MSI. Increased accumulation of amlodipine in APAs treated with amlodipine did indicate direct effects of amlodipine on aldosterone production but further investigations are required for clarification between neoplastic and non-neoplastic aldosterone producing cells.