LipostarMSI is a comprehensive, vendor-neutral software for mass spectrometry imaging (MSI) that supports both untargeted and targeted data analysis. The workflow supported by LipostarMSI covers all the steps required for MSI data analysis: raw data processing, histological co-registration, manual drawing or importing of regions annotated by pathologists, image visualization, a variety of univariate and multivariate image and spectral analysis tools, co-localization analysis, and automated lipids/metabolite annotation, including MS/MS-based identification.