Mass spectrometry imaging (MSI) visualizes the spatial distribution of biomolecules in tissues, whereas ion mobility–mass spectrometry (IM-MS) separates ions through the collision cross-section (CCS) with an inert gas, providing the structural characteristics of isomers. Recent advances have established an integrated workflow, ion mobility–mass spectrometry imaging (IM-MSI), that couples IM with MSI, uniting molecular discrimination with spatial mapping. This synergy has been widely applied in oncology and neuropsychiatric disorders, offering unprecedented insights into biomarker discovery and disease mechanisms. Here, we summarize the principles and classifications of IM-MSI, review their combined biomedical applications, and discuss data processing workflows and commonly used tools.