Abstract In an effort to design a bioassay to screen for biocontrol pathogens and their toxins, procedures were developed to produce calli of yellow starthistle (YST), Centaurea solstitialis (Asteraceae), on solid and liquid media. Three Murashige and Skoog (MS) media with different hormone additives were compared for their effects on the growth of the YST calli. The most effective medium was made of MS salts supplemented with two cytokinins and a low rate of auxin. The growth of the calli slowed when cytokinins were omitted. Similar results were observed in a bioassay setup to compare the reactions of the YST calli with those of detached leaves exposed to potential toxins in the culture filtrates of test fungi. After exposure for 96 h to different concentrations of culture filtrates from three Alternaria alternata isolates, the calli exposed to 100 and 50% concentrations of the filtrates were significantly more damaged than other concentrations tested, based on a visual rating scale. No differences were observed in the damage ratings among the A. alternata isolates tested. An additional bioassay was setup to determine if calli could be used to screen pathogenic fungi. After 48 h, a distinction could be made in a visual rating between calli treated with spores of a pathogen, Phoma exigua, and a nonpathogen, Penicillium sp., confirming that YST calli can be used to screen potential pathogenic fungi against YST.