Theoretical analysis is carried out and a reliable kinetic method for establishment of individuality of cholinesterases in studied preparation is proposed. For reliable conclusion about the presence of single cholinesterase or a mixture of cholinesterases in the biosample, it is necessary to determine values of the experimental kinetic constant of irreversible inhibition by several (three or more) inhibitors, using for evaluation of the catalytic activity of the biomaterial not less than three different substrates consecutively, for example, acetyl-β-methylcholine (substrate selective to typical acetylcholinesterase), butyrylcholine (substrate selective to typical butyrylcholinesterase), and acetylcholine (substrate hydrolyzed easily by cholinesterases of different types).