Despite significant advances in the characterization of asphaltenes, the study of this fraction of crude oil remains challenging for the scientific community and oil companies. It is well-known that asphaltenes are responsible for many of the difficulties found in the extraction, production, transportation, storage, and refining of petroleum. Although they are defined as the fraction of crude oil that is insoluble in paraffinic solvents of low-molecular-weight-type nheptane (C 7 ) or n-pentane (C 5 ), asphaltenes present a great compositional variety, concentrate a variety of elements (among which nitrogen, oxygen, sulfur, vanadium, and nickel stand out), apart from hydrogen and carbon, and are distinguished by grouping the molecules with greater aromaticity of crude oil. Given its high compositional and structural complexity, associating operational problems with specific characteristics of asphaltenes is a task that is still in force. Asphaltenes, in addition to being distinguished by their high aromaticity, are also known for their acid−base properties. The acid character varies according to the origin of the asphaltenes and is related to the presence of carboxylic acids, phenols, carbazoles, and indoles. The basic character can be attributed to the presence of amines, amides, and other nitrogen-containing compounds. Progressing in the detailed compositional characterization of the polar compounds comprising the n-heptane-insoluble fraction (asphaltenes) is still a necessary task toward the understanding of asphaltene aggregation phenomena and the relationship with crude oil properties, such as interfacial tension and viscosity. In the present work, the naphthenic acids of the insoluble fractions of five Colombian crude oils were characterized. The n-heptane-insoluble fraction was subjected to a washing process affording four subfractions, named as extractable 48 h, extractable 72 h, and extractable 96 h, which correspond to the heptane-soluble fractions that were recovered after each indicated time, and asphaltene 96 h, which corresponds to the remaining heptane-insoluble compounds after 96 h of washing. The samples were chosen so that they had a wide range of asphaltene content that varied from 3.0 to 19.2%. The acid fraction was obtained from each sample by solid-phase extraction using aminopropyl silica as an adsorbent material.