Background & Objective: MALDI Mass Spectrometry Imaging (MALDI-MSI) has been used to address clinically relevant questions, but must be performed on Formalin-Fixed Paraffin-Embedded (FFPE) tissues for widespread clinical use. Due to instrument and sample preparation limitations, the spatial resolution falls short of light microscopy. Additionally, the complex sample preparation has raised questions of reproducibility. We present a sample preparation protocol for MALDI-MSI that can distinguish fine structures in FFPE samples, and is reproducible when carried out at different sites. Methods: All sites used a standard operating procedure (SOP) which also covered instruments. FFPE tissues (3-5 µm; mouse intestine, human ovarian teratoma, tissue microarray (TMA) of tumor entities sampled from three different sites) were prepared for MALDI-MSI. Samples were coated with trypsin using an automated sprayer then incubated in a humid environment. After digestion, alpha-cyano-4-hydroxycinnamic acid was deposited using the same sprayer and the section analyzed with a rapifleX MALDI Tissuetyper. After acquisition, statistical analysis and segmentation feature extraction were conducted. Results: Different anatomical regions of the teratoma could be differentiated based on their mass spectrometric profiles and at high spatial resolution, as these corresponded to anatomical structures. Mouse intestine was used to assess whether operators conducting experiments at different sites can obtain similar results. Twenty measurements from two sites exhibited similar peak statistics; ten measurements conducted over five sites and three time points showed reproducible delineation of the villi from underlying muscle. Measurements of a TMA consisting of different tumor samples were used to investigate if different tissue sampling introduced variation. Statistical analysis of the TMA measurements indicated that tissue biology has a greater influence on the spectra than sample origin. Conclusion: These findings indicate that strict adherence to a SOP produces reproducible MALDI-MSI data from FFPE samples and that sampling site is not a major source of variation.