Overall, we successfully developed a highly selective method for the non-target characterization of carboxylic acids in crude oil. The unique fragmentation pathway of carboxylic-BNMA derivatives and coupled LC separation offer a practical approach to simplifying the complexity of HRMS data by targeting a product ion pair, i.e., m/z 169/171. The potential of this approach for different desirable extraction techniques and LC-HRMS systems has been proved through comprehensive optimization and validation in this work. Importantly, the findings contribute to a comprehensive understanding of carboxylic acids in North Sea crude oils, which are essential to petroleum chemistry and environmental studies.