In this study, we show that time of flight-secondary ion mass spectrometry (ToF-SIMS) can be used to detect organic biomarkers, such as hopanes and steranes, in non-fractionated crude oils, without extraction and chemical preparation. Hopanes and steranes may provide valuable information on the history of life on early Earth, particularly if they are present in fluid inclusions in ancient rocks. Due to the presence of different generations of inclusions in even very small rock samples, it would be advantageous to find a method capable of detecting biomarkers in single oil rich fluid inclusions. The capability of ToF-SIMS for detailed chemical analysis of very small sample amounts makes it a potential technique for such analysis, and in this work this possibility is explored. The presence of hopanes and steranes in four different crude oils of different ages and stages of biodegradation was investigated using ToF-SIMS and GC–MS. By combining analyses of biomarker standards, crude oils and chromatographic oil fractions, specific peaks for the different biomarkers were identified in the ToF-SIMS spectra. The presence of these peaks in the spectra from the crude oil samples could be attributed to the biomarkers based on exact mass determination and by comparison with the spectra from the biomarker containing and biomarker lacking fractions, respectively. In addition, the results show that a significant biomarker signal may be obtained from a 10 μm2 oil sample, demonstrating the potential of ToF-SIMS for analysis of single oil bearing fluid inclusions, which in turn may contribute to a better understanding of the early history of life on Earth.