The present study describes an efficient hyphenated flash chromatographic (FC) for the purification of coumarins and polymethoxy flavones (PMF's) from cold pressed grapefruit peel oil (GFO), a byproduct from citrus juice processing plant. GFO was subjected to silica column chromatography using sequential isocratic elution by hexane and chloroform to separate the waxes and phytochemicals enriched fractions, respectively. The chloroform extract was used for hyphenated FC separation using a gradient mobile phase of hexane and acetone. The peak separations were monitored at dual wavelengths of (210)nm and (340)nm. The individual fractions were analyzed by HPLC, pooled and concentrated resulting in isolation of two coumarins (auraptene and bergapten) and three PMF's (tangeretin, heptamethoxyflavone, and nobiletin). The purity and identity of the isolated compounds was confirmed by spectral analysis using high performance liquid chromatography, matrix-assisted laser desorption/Ionization-time of flight-mass spectrometry, and nuclear magnetic resonance. The hyphenated FC separation method was further validated by test of repeatability resulting in low RSD for the yield of the phytochemicals. Utilization of the developed separation method for isolation of value added products from grapefruit byproducts could be beneficial to the citrus processing industry. Supplemental materials are available for this article. Go to the publisher's online edition of Separation Science & Technology to view the supplemental file.