It is often argued that the efficacy of herbal medicines is a result of the combined action of multiple constituents that work synergistically or additively. Determining the bioactive constituents in these mixtures poses a significant challenge. We have developed an approach to address this challenge, synergy-directed fractionation, which combines comprehensive TLC guided profiling with synergy assays for cytotoxicity through brine shrimp lethality study and natural products isolation.