The aim of the work presented herein was to re-evaluate the sample preparation pipeline for mass spectrometry imaging (MSI) experiments focusing on metabolite distributions and drug disposition. The work evaluated the steps from sample collection to quantitative interpretation of the results. A major focus of the work was set on the integration of the evaluated and newly developed workflows with orthogonal tissue imaging techniques. The work evaluated the effects of sample collection in formalin and subsequent preparation into formalin-fixed, paraffin embedded tissues. Overall, these treatments were found to substantially alter the tissue metabolome and distort metabolite and drug distributions, validating the current ‘gold standard’ of fresh-frozen tissues for metabolite and drug disposition focused MSI studies. These high-quality tissues require commonly cryo-sectioning to enable MSI analysis. Sample embedding strategies were explored to allow simultaneous preparation and analysis of several tissue specimens at once to increase technical reproducibility. To achieve highest preservation of the specimens, a novel embedding medium based on a hydroxypropyl-methylcellulose and polyvinylpyrrolidone hydrogel was developed. Within the frame of this work, strategies to decontaminate prepared tissue sections prior to MSI analysis will be reviewed, to minimize the infection risk when handling human tissues or specimen from infection models. Irradiation with UV-C light was found to be a suited decontamination as it enables accurate elucidation of endogenous biodistributions whilst only inflicting minor alterations to the tissue metabolome. The utility of a novel DESI setup based on a triple-quadrupole mass spectrometer was described and its application to elucidate drug disposition within tissues. The quantitative relationship of DESI- and MALDI-MSI were explored and some of the newly developed and established workflows were utilized in a multi-omics approach to elucidate the toxicokinetic effects of polymyxin B1 in a model of drug induced nephrotoxicity.