A supercritical fluid extraction (SFE) method using CO2 has been developed for the removal analysis of fat from ground beef samples for nutritional analysis. The SFE procedure was coupled with a solid phase extraction (SPE) disk for the isolation of fats from acid hydrolyzed ground beef samples prior to SFE. The meat hydrolysate is filtered and collected on the reversed-phase SPE disk which is then subjected to SFE. The extracted fat is then transesterified to fatty acid methyl esters (FAMEs), and total, saturated, and cis-monounsaturated fats were determined by gas chromatography. The SFE method was tested on two commercial extractors and the results compared to a solvent-based (ether/hexane) extraction method. No significant differences were found between the results obtained by the solvent-based and the SC-CO2 methods. The SFE method proved a suitable replacement for a traditional organic solvent extraction methods, thereby eliminating the use and costs associated with solvent disposal as well as the exposure of laboratory personnel to toxic and/or flammable solvents.