Branched chain fatty acids (BCFAs) are one of the important sub categories of fatty acids (FAs) which have unique functions in nature. They are commonly analyzed by GC-MS after derivatization to methyl esters (FAMEs). On the other hand, there is a lack of isomer-selective LC-MS methods which allow the distinction of different isomers with wide coverage of carbon chain length. In this work, a systematic retention and isomer selectivity study on seven commercially available UHPLC columns (six polysaccharide columns Chiralpak IA-U, IB-U, IC-U, ID-U, IG-U and IH-U; one Acquity UPLC CSH C18 column) was performed. Various experimental factors were evaluated including column temperatures, gradient profiles and flow rates to elucidate their effects on the separation ability of homologous series of BCFAs with distinct chain lengths, different branching types and branching positions. In general, IG-U outperformed the other columns in terms of isomer selectivity especially for the short and medium-chain BCFA isomers while RP C18 showed good potential in terms of selectivity for long-chain BCFA isomers. Furthermore, after the evaluation of the chromatographic retention pattern on the various columns and method optimization, we report a methodology for untargeted isomer-selective BCFA profiling without precolumn derivatization with UHPLC-ESI-MS/MS by quadrupole-time-of-flight instrument with SWATH acquisition. The best method provides selectivity for constitutional isomers of BCFAs covering distinct chain length (C5-C20) with different branching types (methyl or ethyl) and branching positions (2Me, 3Me, 4Me, 6Me, anteiso and iso-BCFAs) with an optimized LC condition on Acquity UPLC CSH C18 column. Finally, the optimized method was applied for the BCFAs profiling in lipid extracts of Staphylococcus aureus samples. Besides, pooled human platelets and pooled human plasma were evaluated as mammalian samples for presence of BCFAs as well. The new method showed strong potential for BCFA profiling in bacterial samples including different isomers anteiso and iso-BCFAs, which could be a useful tool for related subdisciplines in metabolomics and lipidomics in particular in combination with electron-activated dissociation MS. Compared to GC, the presented isomer selective LC methods can be also of great utility for preparative purposes. Equivalent (carbon) chain length numbers were calculated for RP18 and Chiralpak IG-U and compared to those of FAMEs obtained by GC.