To fully understand the drug mechanism of action of new chemical entities, pharmacologists need to acquire confident and precise data in pharmacokinetics and in pharmacodynamics and build strong pharmacokinetic/pharmacodynamic relationships. Target engagement in evaluating new chemical entities provides the basis for treatment efficacy. Classical technologies are sometimes limited or inefficient to provide these precise data; however, label-free MS imaging technology is able to provide these molecular features, spatial distributions, quantification and metabolomics data. Important considerations for imaging biological sections are described. Various applications in pharmacology are presented across different therapeutic areas, where MS imaging answers crucial drug discovery and preclinical development needs.