With a rapidly growing number of biomedical applications of mass-spectrometryimaging (MSI) and expansion of the technique into the clinic, spectrum annotation is an increasingly pressing issue in MSI. Although identification of the species of interest is the key to answering biomedical research questions, only few of the hundreds of observed biomolecular signals in each MSI spectrum can be easily identified or interpreted. So far, no standardized protocols resolve this issue.

Present strategies for proteinidentification in MSI, their limitations and future developments are the scope of this review. We discuss advances in MSI technology, workflows and bioinformatic tools to improve the confidence and the number of proteinidentifications within MSI studies.