This paper describes a comparative study of the gravimetric versus hydrolysis/derivatization/gas chromatography-mass spectrometry determination of fat in infant formula. Fat was extracted using supercritical carbon dioxide modified with a small amount of ethanol, the extract was weighed, and the total fat was determined gravimetrically. Subsequently, another sample of the supercritical fluid fat extract was hydrolyzed to yield free fatty acids, which were converted to their methyl ester derivatives (FAMEs). Quantification was performed by GC-MS. NIST Standard Reference Material (SRM-1846) was used to validate both fat determination methods. Results showed that the gravimetric average percent fat was 26.86%, whereas the GC-MS method yielded 24.64%. Some peaks were detected in the ion chromatogram from the GC-MS that were identified as nonfatty acids such as aldehydes, which may account for the higher percentage fat measured as weight of extract rather than measured as FAMEs expressed as triglycerides.