Objectives: The main objectives of this research were to develop a method for blood smear analysis using MALDI TOF MS imaging technology, to improve the ionization of analytes of interest, isolation of individual cell lines and to optimize imaging conditions. Study design: Preclinical development of laboratory procedure. Participants and methods: In this research we have included 4 male participants, voluntary blood donors. After venipuncture, the blood was subjected to the preparation of blood smears according to the three different protocols. Differently prepared samples were applied to ITO slides and analyzed using MALDI TOF IMS. Results: Blood smear prepared according to the first protocol, with the use of ammonium acetate, gives better signals of ATP and heme, but also hemolyzes erythrocytes. Preparation of buffy coat resulted in leukocyte overlap with platelets, which is not suitable for analysis. After the leukocytes isolation, using ammonium chloride, it is not possible to distinguish the types of leukocytes and due to over - processing of the cells, during preparation, the question of the reliability of the results arises. Conclusion: The best ionization of the analyte is given by the laser firing paramters that include laser diameter of 5 μm and the use of a matrix 9-AA contributes to the better targeted signal distribution in the spectra. For the analysis of the chemical composition of erythrocytes, blood smear is the most suitable, but should be less exposed to ammonium acetate, and for the analysis of the chemical composition of leukocytes, the most suitable approach is based on isolation.