The invention relates to medicine and concerns a method for isolating a protein containing insulin and one polysialic acid residue from reaction byproducts and unreacted starting material, by means of reverse-phase chromatography. The method employs the chromatographic material PLRP-S equilibrated with a solution comprising a non-polar component in an amount of 2-8%, and a stepwise gradient elution. The invention provides for a high-purity protein in one isolation step.