Recently, the generation of “knock-out (KO) extracts” has been reported, using affinity chromatography with monoclonal antibodies for removal of single phytoconstituents from plant extracts. Another approach, termed chemical subtraction, uses countercurrent chromatography (CCC) to deplete target(s) from a complex mixture, following the equation: extract (minuend) – target compound (subtrahend)=knock-out extract (difference). Chemical subtraction enables further chemical and biological characterization of both the otherwise intact KO extract as well as the residually complex subtrahend(s). This concept is applied to bioactive prenylated phenols in hops (xanthohumol [XH], isoxanthohumol [IX], and 8-prenylnaringenin [8-PN]), using HSCCC and HEMWat 0 as initial solvent system. Reflecting their different abundance in the extract and differences in CCC selectivity, the subtracted XH (33%), IX (3%), and 8-PN (0.35%) had qHNMR purities of 90, 54, and 12% w/w, respectively. The low 8-PN purity also reflects its co-elution with XH. Thus, a second CCC subtraction was performed using HEMWat -3 and orthogonal HterAcWat +3 systems. All HSCCC fractions other than those of the targets were recombined to provide the KO extracts. Quantitative LC-MS, qHNMR and UHPLC profiles will be presented for four hops KO extracts: XH-KO, IX-KO, 8-PN-KO and XH/IX/8-PN-KO.