Introduction: Mass Spectrometry Imaging (MSI) provides information about the spatial distributions of different analytes on tissue sections and requires no homogenization techniques or labeling. It has a wide spectrum of applications ranging from biomarker discovery to drug response studies. MSI can be adapted to the clinical settings due to its ability to combine mass spectrometry and histological data.Areas covered: The major obstacle to adapt MSI into clinical settings arises from the limited standardization in MSI experiments. We aimed to review the multi-site studies in MSI specifically focusing on reproducibility. Additionally, we emphasized the necessity of quality assessments in MSI for its potential usage in clinical settings.Expert opinion: We discuss how important it is to follow optimized and standardized workflows in MSI and conduct potential quality assessments at important stages in order to adapt MSI into clinical applications.