A set of five oil samples from the same source rock, classified at different levels of biodegradation, was evaluated by high-resolution mass spectrometry (Orbitrap). O-containing acidic compounds from the acidic oil fractions were characterized by determining their molecular formulas. The effects of biodegradation were detected in all O classes and were consistent with the biodegradation of neutral compounds. Additionally, we found a slightly biodegraded sample that contained acidic compounds, biomarkers of a highly biodegraded sample. These results led us to propose a scale of biodegradation using acidic compounds, particularly those containing two oxygen atoms (carboxylic acids).