The present paper attempts to present important issues in the methodology of identifying  lipid and protein changes in brain tissue induced by radiation. The goal was to address the analysis of the methodology and to investigate the feasibility of the generation of lipid/protein profiles of irradiated brain tissue; disregarding the results of the analyses. Lipids and proteins are biomolecules with diverse structures and functionalities that participate in important intracellular processes. Changes in the lipid and the tissue protein profiles may indicate a cellular response to an external stimulus as well as the emergence of neoplasms or neurodegenerative diseases such as Alzheimer's. DESI-MSI is a convenient method for identifying lipids and their spatial distribution in tissue beyond analytical quantification. DESI-MS allows the creation of an image of several low lipid m/z classes. MALDI-MSI has already been a method used in the study of macromolecules as structural, membrane, hormone, neuromediator and immunological peptides. Through a full matrix scan, with a m/z spectrum between 500-1000 for lipids and with a mass spectrum of 1000-15000 Da for proteins, the molecular profiles can be analyzed generating 2D chemical pixel-type images. The produced image allows to associate the distribution of the lipids and proteins in the tissues with their chemical profile identified, allowing the verification of the changes radioinduced. Radiation triggers intense oxidative stress by increasing reactive oxygen species (ROS) and free radicals, causing DNA-damage consequently with alterations in proteomics including cellular lipid and protein expressions. The cellular morphophysiological changes are responsible for both the clonogenic inhibition and the induction of the apoptotic process.  The images´s production was directly dependent on the rigorous execution of the methodological procedures. Innumerable interferences could impair the image´s generations and protein and lipid analysis.  The main interferences in the present MS methodology were addressed and discussed.  Following a well-established methodology, DESI can  generate lipid and MALDI protein spectra in brain tissues. Near future, it will be possible to detect changes of  the spatial distribution and the types of lipids and proteins in the white and gray matter regions.