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Matrix-assisted laser desorption/ionization imaging high-resolution mass spectrometry (MALDI-imaging-HRMS) is an important technique for visualizing the spatial distribution of compounds directly on the surface of organisms such as microorganisms, insects, plants, animals, and human tissues. However, MALDI-imaging-HRMS and the stable isotope labeling approach have never been combined for the detection and simultaneous visualization of labeled and unlabeled compounds, their analogues and derivatives, as well as their precursors. Herein, we present a methodology that labels microbial secondary metabolites directly on agar with stable isotopes and allows concurrent spatial distribution analyses by MALDI-imaging-HRMS. Using a thin film of labeled agar supplemented with [1-13C]-l-proline, [methyl-D3]-l-methionine, 15NH4Cl, or [15N]-l-serine overlaid on unlabeled agar, we demonstrate the incorporation of labeled precursors into prodiginines and serratamolides produced by an endophytic bacterium, Serratia marcescens, by MALDI-imaging-HRMS and HPLC–HRMS. Further, we show the incorporation of CD3into prodigiosin as well as its characteristic fragments directly by MALDI-imaging-HRMS2. Our methodology has several advantages over currently existing techniques. First, both labeled and unlabeled compounds can be visualized simultaneously in high spatial resolution along with their labeled and unlabeled precursors. Second, by using a thin film of labeled agar, we utilize minimum amounts of expensive labeled compounds (1–3 mg) ensuring a cost-effective method for investigating biosynthetic pathways. Finally, our method allows in situvisualization and identification of target and nontarget compounds without the need of isolating the compounds. This is important for compounds that are produced by microorganisms in low, physiologically, or ecologically relevant concentrations.