Normal-phase high pressure liquid chromatography with isocratic elution on Lichrosorb Diol Columm has been used to separate and quantify androstendione, testosterone, 2-α-hydroxytestosterone, 6-β-hydroxytestosterone, 16-α-hydroxytestosterone and 7-α-hydroxytestosterone. These steroids are the major products of testosterone hydroxylation by rat liver microsomes. Separation was achieved in 25 minutes by using and isocratic mixture of n-hexane: 2-propanol: 9:1 (v/v). This method compares very favorable with other methods published to study microsomal hydroxylations in different positions of the steroid skeleton.