Licorice is the most frequently prescribed herb in traditional Chinese medicine (TCM) and Japanese Kampo medicine. Although glycyrrhizin (GC) is one of the major active components of licorice, the other components are also known to possess biological activities. We had previously prepared GC-knockout (GC-KO) extract, which contained all components of licorice extract (LE) except GC using a immunoaffinity column conjugated with anti-GC monoclonal antibodies (MAb). In this study, we used the GC-KO extract to investigate the potential role of GC in LE-induced apoptotic cell death. LE markedly induced apoptotic cell death in human leukemia HL-60 cells. Although treatment with GC alone had no influence on cell viability, induction of cell death by the GC-KO extract was weaker than that by LE. Interestingly, the addition of GC to the GC-KO extract significantly rescued cell growth inhibition and activated caspase-3. These data suggest that GC may synergistically enhance the induction of apoptosis in the presence of other components of LE. Moreover, these pharmacological analyses indicate the utility of KO extracts in determining new functions of target compounds and the interactions between target compounds and other constituents of medicinal plant extracts.