Research regarding the distribution of metabolites is a vital aspect of insect molecular biology. However, current approaches (e.g., liquid chromatography-mass spectrometry or immunofluorescence) have cons like requirement of massive tissues, low efficiency, and complicated operating processes. As an emerging technology, mass spectrometry imaging (MSI) can visualize the spatiotemporal distribution of molecules in biological samples without labeling. In this chapter, we retrospect the major types of in situ measurement by MSI, and the application of MSI for investigating insect endogenous and exogenous metabolites and monitoring the dynamic changes of metabolites involved with the interactions between insects and plants. Future studies that combine MSI with other genetic tools can facilitate to better explore the underlying mechanisms concerning insect physiology and metabolism.