Polylysine is a small natural homo-polymer made out of amino acid lysine that is produced by bacterial fermentation. Due to the fact that it is a complex mixture of polymers with different molecular weight it is very hard to obtain a single sharp peak for this compound. Polylysine was successfully analyzed on a Primesep AP column in HILIC/cation-exclusion mode. Cation-exclusion mode on a Primesep AP column allows to obtain sharper peak. Presence of the basic groups on the surface of silica eliminated silanol interactions between basic sites of polylysine and acidic sites of Primesep AP column.