An aseptic bioassay utilizing female and male donor materials was performed in order to assess the allelopathic activities of cladophylls and root systems in dioecious asparagus. The aseptic sandwich method revealed that the growth of acceptor plants was significantly inhibited by the addition of over 30 mg fresh weight of cladophylls. When female and male cladophylls were separately adopted for the sandwich method, there was no sexual difference in the allelopathic activity of both youthful and senescent cladophylls. To detect the allelopathic effects of root systems, the plant box method employing female and male regenerants was used aseptically, and the growth of acceptor plants was seriously inhibited irrespective of sex expression in donor plants. Early in vitro flowering seedlings expressing individual sex were effectively used for the bioassay, and the results indicated that the degree of allelopathic activities was the same as that in regenerants. These results show that allelopathic activity in female and male plants was equivalent, and that the application of materials that can distinguish between the sexes would be useful for assaying the mutual effects of sexuality on allelopathy. The aseptic bioassay system that employed female and male materials presented here could promote the isolation and identification of allelochemicals.