In situ combustion (ISC) is one of the highest potential enhanced oil recovery (EOR) processes for heavy oils. However, several operational issues, including the formation of highly stable emulsions, have limited its application. Disclosing the physicochemical proprieties of these emulsions, especially the chemical nature of the compounds involved in the stabilization process, has become relevant for the success of ISC projects. In the present work, the physicochemical changes at a laboratory-scale low-temperature oxidation (LTO) regimen performed over a Colombian heavy crude oil were followed by mass spectrometry. The compositional analyses were performed using both positive-ion atmospheric pressure photoionization ((+) APPI) and negative-ion electrospray ionization ((−) ESI) Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS). Further isolation of acidic compounds and surface-active species allowed us to determine that the process incorporates a wide variety of compounds to build up the O/W (oil/water) interface, thus increasing the stabilizing tendency of the emulsions. During the combustion, oxygen is chemically incorporated to the crude over hydrocarbon compounds, as well as over sulfur- and nitrogen-containing compounds, generating classes such as O, O2, O3, O4, OS, NO2, and NO3 that explain the high viscosity and high stability of the emulsions.