Carboxylic acids play an essential role in petroleum chemistry. Due to their amphiphilic nature, carboxylic acids in are surface active and contribute to the interfacial behavior and stability of water-oil emulsion systems, which is critical in production systems. The acidic fractions are associated with catalyst poisoning and corrosion of equipment and pipelines, leading to problems in production and refining. However, carboxylic molecules are also valuable markers providing important information about the migration, maturation, and degradation of petroleum. Furthermore, due to their toxicity, aromatic and naphthenic acids are of environmental concern. A detailed characterization of carboxylic acids in crude oil is therefore desirable but remains a significant challenge. It is well-recognized that their structural and compositional diversity and the complexity of crude oil matrices are analytically challenging. Several attempts have been made to separate and enrich carboxylic acids from petroleum samples for further analysis. Several studies employed liquid-liquid extraction (LLE) due to its simplicity and convenience. Other approaches have been explored to improve selectivity and efficiency, including (micro) solid-phase extraction, magnetic dispersive extraction, and ionic liquid extraction . Analysis without prior sample treatment using high-resolution mass spectrometry (HRMS), including Orbitrap and Fourier-transform ion cyclotron resonance mass spectrometry (FTICR-MS), allows determining a large number of compounds via direct infusion due to the high resolving power and mass accuracy. High levels of heteroatomic compositional detail are acquired accordingly, including the molecular composition, elemental distribution, degree of saturation, and abundance. However, these methods do not provide molecular structures or connectivity. Thus, despite the instrumental advantages and improvements of direct infusion HRMS, hyphenated techniques are beneficial to increase structural coverage and information. Therefore, recent method developments have focused on HRMS coupled with liquid and or gas chromatography, including multidimensional separations. Ultimately, developing new, more effective analytical strategies within both sample preparation, analytical separations and data processing is essential to increase our understanding of petroleum acids.