A reversed-phase HPLC-tandem mass spectrometry (RP-HPLC-MS-MS) method was refined for the positional analysis of complex mixtures of TAG. This method has the advantages of speed, ease of automation, and specificity over traditional digestion-based methods for the positional analysis of TAG. Collision-induced dissociation (CID) of ammoniated TAG in an ion-trap mass spectrometer produced spectra that were dependent on the FA position. Dominant DAG fragments were formed from the loss of a FA moiety from the ammoniated TAG species. The loss of FA in the outer positions was favored over their loss in the central position. The combination of RP-HPLC and CID produced spectra that were free of the isotope effects that can complicate spectral interpretation in existing methods. The combination also provided selectivity based on the chromatographic fractionation of TAG, in addition to the selectivity inherent in the CID process. Proof-of-concept experiments were performed with binary mixtures of TAG from the SOS/SSO, OSO/OOS, and the PSO/POS/SPO positional isomer systems (where S is 18∶0, stearic acid; O is 18∶1 (cis-9), oleic acid; and P is 16∶0, palmitic acid). Plots of fractional DAG fragment intensities vs. fractional composition of the binary mixtures were linear. These plots were used to determine the fractional composition of each of these isomeric systems in a variety of vegetable oils and animal fats. Current limitations, future developments, and applications of this method are discussed.