The nutrient composition of asparagus (Asparagus officinalis L.) depends on the harvest period. We visually determined the changes in the relative amounts of asparaptine A and rutin in whole asparagus samples for different harvest periods (May and July) using matrix-assisted laser desorption/ionization (MALDI)–imaging mass spectrometry (IMS). The whole-tissue localization of these functional compounds was determined using longitudinal sections. Asparaptine A was localized throughout the ear tip and stalk regions, and its level was greater in the July harvest period than in the May harvest period. Rutin was localized at the epidermis and exhibited a higher concentration in the early harvest of May, providing protection from UV radiation. For rutin, the relative intensity values of IMS data were in good agreement with the high-performance liquid chromatography (HPLC) data. The combined evaluation of IMS and HPLC has great potential for visually and quantitatively investigating the effects of the harvest period on the nutrient composition and localization and for enhancing commodity values.