In this study, we focus on isolation and fractionation strategies by solid phase extraction (SPE) for a broad range of environmentally related organic acids. These emerging potential contaminants are primary degradation products of spilled petrogenic compounds but little attention has been given to their environmental analysis and risk assessment. Three mixed-mode anion exchange sorbents possessing various backbone polarities were compared with respect to the extraction and fractionation efficiency. Quantitative analysis was obtained using an optimized and validated LC-MS method. We demonstrated the importance of reasonable ion source settings for highest sensitivity of individual carboxylic acids. Furthermore, fractionation of carboxylic acids into aliphatic and aromatic fractions by SPE was achieved by step-wise elution. Best performance regarding the isolation of low-concentration acids (sub μg L ⁻¹ level) in complex samples was attributed to Strata XA and is caused by high secondary hydrophobic interactions in addition to the ionic interactions. Isolation and fractionation of acid spiked diesel-spilled harbor water (22 μg L ⁻¹ spike level) revealed recoveries >80% for all tested carboxylic acids and low carry-overs of neutral petroleum compounds. We conclude that the isolation and fractionation of acidic degradation products by SPE is a powerful tool to investigate this new class of contaminants in relation to identification and ecological risk assessments.