Rationale Sample preparation is one of the most crucial steps for matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI). Scientists beginning their study with this technique may be overwhelmed by the variety of matrices, solvents, and concentrations; the methods of their applications; and the lack of widely available knowledge of the effect of these parameters on the results. Here we present in depth the aspects of matrix deposition, which will be helpful for the scientific community. Methods In this study, we tested several MALDI matrices, such as 2,5-dihydroxybenzoic acid (DHB), norharmane, N-(1-naphthyl)ethylenediamine dihydrochloride (NEDC), and 9-aminoacridine (9AA), using the SunCollect system: wet-interface matrix deposition in the context of lipid analysis. We optimized the number of matrix layers and nozzle settings in terms of spectral intensity and the overall quality of the obtained ion maps. Results Our research presents the effect of the number of matrix layers and nozzle settings on the results and allows for choosing the optimal parameters for the analyses. In positive ionization mode, DHB matrix could be chosen first. In the negative ionization mode, 1,5-diaminonaphthalene matrix produces a higher peak intensity in a lower mass range and seems to provide more information than 9AA. We recommend NEDC for particular processes such as glucose analysis. Compared to the remaining matrices, norharmane shows significant changes in the obtained ion maps. Conclusions Such a large amount of data allow us to observe an interesting conclusion: the obtained ion image for a particular ion could differ dramatically with a change in the matrix, the solvent composition, or even the number of matrix layers. This must be considered when interpreting the result, impelling us to compare the results obtained with different matrices with caution.