Abstract: Method of preparation of restriction endonucleases, particularly those exhibiting the desired sequential specificity consists in that a fluorescence-marked DNA probe is used for screening a library of mutants, preferably in IVC format, and/or using other high-performance screening (HTS) technique, which is attained through expression of proteins included in the library of mutants in a cell-free system in the presence and by means of the DNA probe, and proteins thus obtained, resulting from expression of clones from the library, degrade the DNA probe, if their substrate specificity matches the searched one, the degradation of the DNA probe being detected as a disappearance of the FRET phenomenon between fluorescence markers included in the probe, and then microcompartments in which the FRET phenomenon ceases to occur, are separated from the remaining ones using Fluorescence Activated Cell Sorter (FACS) and/or other equipment for HTS analysis, and then DNA coding clones capable of degrading the probe are amplif