An aseptic bioassay system to estimate allelopathy in asparagus calluses was established. Allelopathic activities of calluses were measured according to the callus packed cell volume (PCV) by means of the sandwich method, and remarkable inhibition of acceptor plants was observed by the addition of over 0.1 mL PCV. When female and male calluses were separately used for the sandwich method, there was no sexual difference in allelopathic activity. To detect the allelopathic activities of the callus cells in detail, we improved the plant box method that substituted the calluses for the seedlings, and serious inhibition of acceptor plants was observed with the addition of over 0.35 mL PCV as donor material. Allelopathic activities were accelerated by irradiating the calluses with light-emitting diodes (LEDs) prior to performing the aseptic bioassay. It also seemed that the light quality would affect their activities. The allelopathic activity appeared intensely with irradiation of the calluses with a blue LED. The aseptic bioassay system that employed LED-irradiated calluses established here could accelerate effectively the isolation and identification of allelochemical(s).