Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) is commonly used in food and pharmacological sciences to visualize localization of drugs and food compounds and their metabolites in plant, animal, and human tissues. The localization of compounds obtained by MALDI-MSI images provides useful information for elucidating their physiological and pharmacological properties. Food polyphenols, naturally occurring in tea, coffee, fruits and vegetables, have health benefits owing to their preventative effects against conditions such as cancer, diabetes, and cardiovascular diseases. In order to elucidate the pharmacological properties of polyphenols, their absorption, distribution, metabolism and excretion must be investigated. However, application of MALDI-MS imaging for polyphenols is challenging due to lack of an appropriate matrix reagent to visualize polyphenols in targeted biological tissue. The present work highlights the development of MALDI-MSI for visualization of food polyphenols. Nifedipine, which produces a nitrosophenyl pyridine derivative under laser irradiation, could be a new matrix for MS detection of polyphenols. The combination of nifedipine and phytic acid (a metal-chelating agent) successfully achieved MS visualization of polyphenols in biological tissue. The inhibitor-aided MALDI-MSI has been applied for elucidation of intestinal absorption routes and metabolic behaviors of polyphenols. The MALDI-MSI technique shows great potential for visualizing absorption, distribution and metabolism processes of food polyphenols.