The use of matrix laser desorption/ionization mass spectrometry (MALDI) for the study of small molecules is still critical and limited by the efficiency of the matrices. In this work, for the first time, the effect of anion and cation in the structure of ionic liquids to obtain high-intensity MALDI spectra of amino acids is shown. Ten ionic liquids based on substituted imidazole were tested. Three amino acids (histidine, alanine and serine) were used as test compounds to assess the effectiveness of ionic liquids. It was found that the BMIMC6F5BF3 ionic liquid, used for the first time as a MALDI matrix, showed the best characteristics in terms of ionization efficiency for the analytes under consideration. Mass spectrometric imaging was used to estimate the degree of homogeneity in the distribution of the analyte over the surface and in the volume of the matrix with ionic liquid. It has been shown that, without ionic liquid, approximately 40% of the total surface area with an extremely low amino acid content is observed on the matrix surface. Whereas in the presence of ionic liquid there are only 3% of such areas. The use of an ionic liquid allows the analyte to be concentrated in the surface layer of the matrix. At the same time, without IL, only when using a classical matrix, there is an uneven distribution of amino acid both in the surface layer and throughout the entire volume of the matrix.