<p>Acylcarnitines (ACs) are metabolic intermediates of fatty acids playing important roles in regulating cellular energy and lipid metabolism. The large structural diversity of ACs arises from variations in acyl chain length and the presence of chemical modifications, such as methyl branching, desaturation, hydroxylation, and carboxylation. Numerous studies have demonstrated that these structural isomers of ACs function as biomarkers for a variety of diseases. However, conventional tandem mass spectrometry (MS/MS) via low-energy collision-induced dissociation (CID) faces challenges in distinguishing these isomers. In this study, we report a radical-directed dissociation (RDD) approach for characterization of the intrachain modifications within ACs. The method involves derivatizing ACs with O-benzylhydroxylamine (O-BHA), followed by MS² CID to produce a nitroxide radical for subsequent RDD along the fatty acyl chain. The above RDD approach was employed on a cyclic ion mobility spectrometry (cIMS) and reversed-phase liquid chromatography (RPLC), enabling the identification and relative quantification of branched chain isomers of ACs. By derivatizing carboxylated ACs with O-BHA, their mass is shifted to a higher region, thereby facilitating their separation from the isobars of hydroxylated ACs. Furthermore, this RDD method effectively allows for the assignment and localization of C = C and hydroxylation positions. This RDD approach has been applied for in-depth profiling of ACs in mice plasma extracts.</p> Graphical abstract <p></p>