The strength of matrix-assisted laser desorption/ionisation-mass spectrometry imaging (MALDI-MSI) is to analyse and visualise spatial intensities of molecular features from an intact tissue. The distribution of the intensities can then be visualised within a single tissue section or compared in between sections, acquired consecutively. This method can be reliably used to reveal physiological structures and has the potential to identify molecular details, which correlate with biological outcomes. MALDI-MSI implementation in clinical laboratories requires the ability to ensure method qualification and validation to meet diagnostic expectations. To be able to get consistent qualitative and quantitative results, standardised sample preparation and data acquisition are of highest priority. We have previously shown that the deposition of internal calibrates onto the tissue section during sample preparation can be used to improve mass accuracy of monitored m/z features across the sample. Here, we present the use of external and internal controls for quality check of sample preparation and data acquisition, which is particularly relevant when either a large number of spectra are acquired during a single MALDI-MSI experiment or data from independent experiments are processed together. To monitor detector performance and sample preparation, we use egg white as an external control for peptide and N-glycan MALDI-MSI throughout the experiment. We have also identified endogenous peptides from cytoskeletal proteins, which can be reliably monitored in gynaecological tissue samples. Lastly, we summarize our standard quality control workflow designed to produce reliable and comparable MALDI-MSI data from single sections and tissue microarrays (TMAs).