Abstract Background The development of MALDI Imaging Mass Spectrometry is a promising technique in the investigation of biological molecular repertoire. We have pursued an analytical assessment of this technique in its application to proteome analysis. Methods A specific statistical method of analysis has been developed to enable data processing in the absence of internal standards, by defining similarity scores. Results The investigated linear mode MALDI-TOF set-up allows to obtain data variations comprised within the 30% of variation when assaying tissues samples from the same animal, while the 60% of variation was highlighted in the inter-mice series assaying syngenic animals. Conclusions This analytical assessment represents the first step of a process that should validate the utilisation of this technique in the clinical practice.