Four phenylbutanoids, (E)-4-(3,4-dimethoxyphenyl)but-3-en-l-ol (I), (E)-4-(3,4-dimethoxyphenyl)but-3-en-l-yl acetate (II), (E)-1-(3,4-dimethoxyphenyl)butadiene (III) and (E)-3-(3,4-dimethoxyphenyl)-4-[(E)-3,4-dimethoxystyryl]cyclohex-1-ene (IV), isolated from Zingiber cassumunar, were used as standard markers for quantitative determination and preparation of phenylbutanoid-enriched Z. cassumunar extracts (PZEs). A reversed-phase HPLC method was established for the simultaneous determination of the phenylbutanoids in Z. cassumunar extracts. Systematic extraction studies to maximize phenylbutanoid content revealed that hexane was the most appropriate solvent for extraction. A one-step purification of the hexane crude extract of Z. cassumunar, using silica gel vacuum chromatography, provided the PZEs. The content of phenylbutanoids in the PZEs was up to 48.3% w/w dry weight. The anti-inflammatory activity of PZEs via inhibition of nitric oxide production by murine macrophage-like RAW264.7 cells was stronger than those of the four individual phenylbutanoids, the crude hexane extract and the essential oil of Z. cassumunar. Copyright © 2012 John Wiley & Sons, Ltd.