Abstract: Glycerides (Glys), a class of esterification products of glycerol and fatty acids, serve as significant pathological markers for a variety of metabolic diseases. From the structural perspective, the different linkages (straight- or branched-chain), C＝C bond positions, and other factors of fatty acyl chains lead to the existence of numerous isomers in Glys, and the key to achieving isomeric differentiation lies in the structural identification of the fatty acid chain. In the present research, taking a group of monoacylglycerol (Mag), diacylglycerol (Dag), and triacylglycerol (Tag) with the same fatty acyl structure as examples, a novel mass spectrometry (MS) method for identifying fatty acid chains was proposed, which was achieved by comparing the consistency of the fatty acyl chain cations generated by collision-induced dissociation (CID) of Mag, Dag, and Tag with the quasi-molecular ions of known fatty acids. Quadrupole-time-of-flight tandem MS was utilized to obtain high-resolution m/z values of precursor and fragment ions for glycerin monostearate (Gms), glycerin distearate (Gds), glycerol tristearate (Gts), stearic acid (Ste), and isostearic acid (iSte). To comprehensively record the CID behaviors of the target ions, full collision energy ramp-MS² spectra (FCER-MS²) of Ste and iSte cations, as well as full collision energy ramp-pseudo-MS³ spectra (FCER-pseudo-MS³) of the Ste-unit cations generated by the in-source CID of Gms, Gds, and Gts, were constructed based on online energy-resolved MS. The differential fragmentation patterns of isomers were investigated by combining MS fragmentation rules and quantum chemical calculation. MS² spectra of Ste and iSte exhibited strong similarities, characterized by a series of fragment ions with a consistent mass difference (Δ) as 14 u. However, the FCER-MS² spectra of the two were distinguishable, such as the collision energy level corresponding to 50% survival yield (CE₅₀) of the ion at m/z 285.3 and the optimal collision energies (OCEs) of ions at m/z 87.1, 75.1, and 51.1. As expected, the FCER-pseudo-MS³ spectra of Ste-unit cations from Gms, Gds, and Gts were essentially consistent with the FCER-MS² spectrum of Ste cation, but significantly differed from the FCER-MS² spectrum of iSte cation. Consequently, it is feasible to identify the fatty acyl chain of Gly by comparing the FCER-pseudo-MS³ of fragment ions-of-interest with the FCER-MS² of the candidate fatty acid ions. This approach provides a viable method for identifying structural units of other complex compounds.