A method of instituting hydrolysis by using enzymes of the examined liver is proposed as an adjunct to the formerly elaborated fluorometric procedure of determining folacin in the liver. The fermentative hydrolysis is conducted in two stages. The purpose of the first of them is to split the combined form of folacin under the effect of the examined liver conjugases; the second stage involves transformation of xanthopterin contained in the liver into leucopterin by xanthinoxidase of the liver. When utilizing enzymes of the examined liver the need for producing and application of the enzymatic preparation made of the pig kidneys falls away, this simplifying the fluorometric determination of folacin in the liver and increasing its accuracy.