Mass spectrometry imaging (MSI) allows us to visualize molecular information within samples from biological samples. In this protocol we provide the experimental details for sample sectioning, matrix application, data analysis and histochemistry for the use with an atmospheric pressure (AP)-SMALDI MSI source. This protocol is intended for tissue sections, allowing to obtain MS images for these samples with down to 1 µm lateral resolution. Sample sectioning and matrix application are key steps for a successful tissue analysis and therefore the experimental steps are described in detail. As example, lipids were imaged in mouse brain tissue section with a step size of 10 µm using the AP-SMALDI MSI source. The data analysis, including MS image generation and the data base procedure, which is based on accurate mass measurements, are further described and presented in the protocol. MS image analysis for this experiment shows an excellent correlation with post-MSI histological staining. Compound assignment is based on accurate mass measurements of less than 2 ppm mass error with high mass resolution (R=140.000 at m/z=200) for identification of analytes in tissue sections.