<p>Enzymes play essential roles in numerous biochemical reactions, making it necessary to evaluate both their localization and activity. While conventional enzyme activity detection methods require specific antibodies and colorimetric reactions for each target compound, a recent method using mass spectrometry imaging enables the visualization of enzyme distribution and activity by applying substrates directly to tissue sections and detecting the resulting enzyme reaction products generated on these sections. Notably, the 11β-hydroxylase (CYP11B1, EC 1.14.15.4) reaction plays a key role in stress response, metabolism, and immune function regulation. Consequently, the regulation of CYP11B1 activity can significantly impact these physiological processes, enabling improved drug development. Therefore, controlling this enzyme activity will facilitate the development of new therapies for related diseases. This study investigated a method for visualizing the distribution and relative activity of CYP11B1 in cortisol-producing adrenocortical adenoma (CPA) tissue sections derived from patients with mild autonomous cortisol secretion (MACS). By optimizing key parameters such as the substrate and coenzyme concentrations, together with reaction times, the method successfully revealed the relative CYP11B1 activity in human MACS. Additionally, visualization was achieved in aldosterone-producing adrenocortical adenoma (APA) derived from patients with primary aldosteronism and in non-functioning adrenal adenoma (NFA) derived from patients with adrenal cortical adenoma. The relative CYP11B1 activities of CPA, APA, and NFA samples obtained in this study correlated with the clinical information and were consistent with the previously reported cortisol, adrenocorticotropic hormone, and dexamethasone suppression test profiles in blood and urine, thereby validating the proposed method.</p> Graphical abstract <p></p>