An analytical procedure is presented for obtaining detailed characterization of petroleum hydrocarbons which undergo microbial degradation. The procedure includes column chromatographic separation and characterization of the resulting fractions by mass spectrometry and gas chromatography. The use of computerized low-resolution mass spectrometry is offered as a method for assessing microbial degradation of petroleum. This method provides information which cannot, at the present time, be obtained by other available analytical methods. Use of this method to evaluate degradation of a South Louisiana crude oil by a mixed culture of estuarine bacteria revealed that asphaltenes and resins increased by 28% after degradation, while saturates and aromatics decreased by 83.4% and 70.5%, respectively. Most of the normal and branched-chain alkanes were degraded (96.4%), but an increase in long-chain alkanes (C28–C32) after degradation was observed by gas–liquid chromatography. Susceptibility of cycloalkanes to degradation was less as the structure varied, i.e., 6-ring > 1-ring > 2-ring > 3-ring > 5-ring > 4-ring. Susceptibility of aromatic components to degradation decreased with increase in the number of rings, viz., monoaromatics > diaromatics > triaromatics > tetraaromatics > pentaaromatics. Aromatic nuclei containing sulfur were twice as refractory as non-sulfur analogs.