A method is described for the production of bile salt derivatives via the substituent hydroxyl groups on the steroid nucleus that permits derivatization of taurine and glycine conjugates. The method involves the oxidation of the hydroxyl groups at C-3 alpha or C-7 alpha on the nucleus by the action of hydroxysteroid dehydrogenases. The keto bile salts produced are isolated and reacted with 2,4-dinitrophenylhydrazine or Dns-hydrazine to yield ultraviolet absorbing or fluorescent derivatives, respectively. The bile salt hydrazones were separated using a reversed-phase C18 radial compression cartridge with a methanol-phosphate buffer gradient elution. Although the 3-keto derivatives of chenodeoxycholic and deoxycholic acids were not resolved, the 7-keto Dns-hydrazone of chenodeoxycholic acid was separated from 3-keto derivative of deoxycholic acid.