The ratio of bile acids in the bile of rats with alloxan diabetes was investigated using the method of thin-layer chromatography. Changes of coefficients of conjugation and hydroxylation of bile acids were calculated and analyzed in half-hour samples of bile obtained during the 3-hour experiment. It has been found that the processes of conjugation of cholic acid with glycine and taurine are inhibited in alloxan diabetes. At the same time a significant increase of free threehydroxycholic and dіxydroxycholic bile acids and conjugates of the latter ones with taurine has been registered. Coefficients of hydroxylation іn alloxan diabetes show the domination of “acidic” pathway in bile acid biosynthesis that is tightly connected with the activity of mitochondrial enzymes.