A method is described for the determination of pregnanediol in human pregnancy urines which have been stored for 2 years under butanol. Hydrolysis of the pregnanediol conjugate is carried out in presence of toluene to protect the liberated pregnanediol. The pregnanediol is purified by combining the method of Sommerville and Marrian with a petroleum ether digestion and, if necessary, with the NaOH-treatment of Astwood and Jones. Final estimation of the pregnanediol may be gravimetric or colorimetric.