Abstract: The determination of the essential metabolite 5β-H-17-OH-Androstane (5β-MT) from methyl testosterone (MT) in rabbit blood by gas chromatography-mass spectrometry was performed to supply method and data for pharmacokinetics study. 2mL blood taken from six rabbits ear was collected at setting time point before and after P.O. administration 20mg MT,1mL plasma sample was extracted with methanol, then the extractant was enzymed by β-glucuronidase. Quantitative analysis was performed with internal standard DHMT. The result showed that 5β-MT was presented in each blood sample, the maximal average mass concentration of six rabbit is 2.2mg/L at 3h.