Mass spectrometry imaging has advanced from a niche technique to a widely applied spatial biology tool operating at the forefront of numerous fields, most notably making a significant impact in biomedical pharmacological research. The growth of the field has gone hand in hand with an increase in publications and usage of the technique by new laboratories, and consequently this has led to a shift from general MSI reviews to topic-specific reviews. Given this development, we see the need to recapitulate the strengths of MSI by providing a more holistic overview of state-of-the-art MSI studies to provide the new generation of researchers with an up-to-date reference framework. Here we review scientific advances for the six largest biomedical fields of MSI application (oncology, pharmacology, neurology, cardiovascular diseases, endocrinology, and rheumatology). These publications thereby give examples for at least one of the following categories: they provide novel mechanistic insights, use an exceptionally large cohort size, establish a workflow that has the potential to become a high-impact methodology, or are highly cited in their field. We finally have a look into new emerging fields and trends in MSI (immunology, microbiology, infectious diseases, and aging), as applied MSI is continuously broadening as a result of technological breakthroughs.