Abstract: A simple method for determining androgens and anabolic steroids in human urine by HPLC has been developed. The compounds studied are nandrolone, methandienone, testosterone, methyltestosterone, testosterone propionate and nandrolone phenylpropionate. The stationary phase used is Cs bonded silica. Isocratic elution was done with CH₃OH—CH₃CN—H20 (4:5:6) and programmed flow Detection limit can be less than 1 ng at the wavelength of 254 nm. The standard curves for each steroid have been set using internal standard (progesterone) and peak height ratio. Linear relationship exists between the ratio and concentration for each steroid. High and stable recovery has been achieved using Sep-Pak C₁₈ cartridges for urine sample clean-up. The operation of the method is simple. The enzymatic hydrolyzation of conjugated steroids in human urine has also been investigated.