Cover crops are defined as crops grown primarily for agroecosystem improvement rather than for market or sale. The use of cover crops to decrease the negative effects of weeds and improve soil and ecosystem health is increasing, but unanticipated allelopathic responses to those cover crops by subsequent crops is sometimes a problem. Allelopathy is broadly defined as the biochemical interactions between all types of plants, including microorganisms. Because allelopathic effects include both inhibitory and stimulatory responses and may be species- and cultivar-specific, a method of rapidly screening donor and recipient crops for allelopathic interactions is needed. The objective of this research was to evaluate a growth chamber bioassay for rapidly screening spinach (Spinacia oleracea L.) cultivars for allelopathic interactions with an aqueous extract from fresh whole-plant tissue of sorghum-sudangrass [Sorghum bicolor (L.) Moench; SSG] cultivars. The bioassay exposed the seed of 10 spinach cultivars to the aqueous extract of three cultivars of SSG during the imbibition and germination processes and evaluated the consequent root and stem development. Compared with the control, the extract from all three SSG cultivars decreased the root length of all spinach cultivars. A subsequent field screen where spinach cultivars were planted into decomposing SSG residue resulted in a similar pattern of growth suppression. These results demonstrate that the growth chamber bioassay is suitable for predicting allelopathic interactions between cultivars of SSG and cultivars of spinach and can be used by growers for making cultivar selection decisions when spinach follows SSG in a cropping sequence. This rapid-screening growth chamber bioassay protocol eliminates many of the environmental and other challenges frequently associated with field trials and may be adaptable for predicting allelopathic interactions among other cover crops, weeds, and subsequent market crops.