For the assessment of androgenesity in vivo urinary 17-KS has often been mesured so far, but the significance of measuring 17-KS still poses many problems. On the other hand, the assessment of androgenesity on the basis of urinary testosterone is direct and superior, but on account of the close polarity of 17-KS fraction and gestagen on chromatography as well as because they are steroids secreted only in a minimal quantity, the only available method reported is to isolate them in three steps at least by paper chromatography or thin layer chromatography. In view of this, the author devised a new method to assay urinary testosterone. This method uses two-step hydrolysis; first β-glucuronidase hydrolysis and solvolysis. Then the extraction (1) with ethylacetate is carried out. Next, the isolation (2) is conducted by column chromatography, and the separation (3) by impregnant thin layer chromatography. After scraping off the separate, it is oxidized with chromic acid (4) and finally, the qualitative analysis is done by the coloration method of Zimmerman. By this method of isolation and qualitative analysis of testosterone from urine samples fairly satisfactory results were obtained. The results have demonstrated that the pattern of urinary testosterone during normal menstrual cycle shows its peak before and after ovulation as well as immediately before menses. In addition, testosterone in the urine also exists in the form of a solvolysate, and its pattern during normal menstrual cycle and its alerations on taking drugs show behaviors often different from those of β-glucuronidase hydrolysate.