The human gut microbiome plays a crucial role in both health and disease. Metabolites in human faeces related to microbial activity might therefore be attractive surrogate markers to track changes of microbiota induced by diet or disease. The hyphenation of gas chromatography with triple quadrupole mass spectrometry is a promising approach to increase sensitivity and selectivity as compared to single quad MS instruments. The versatility of gas chromatography-tandem mass spectrometry (GC-MS/MS) can be advantageously exploited in clinical laboratory medicine, e.g. for quantification of sterols in biological material. In this paper, we present the application of GC-MS/MS for determination of sterol components in human faeces. A serious problem of analysis of faeces is preanalytics. Uncontrolled degradation of metabolites during transport and storage of faeces before entering the clinical laboratory might occur. In our experiments we did not observe any increasing or decreasing concentration after storage of native faeces material even at room temperature. Furthermore, we answer the question of how personal metabolic responses with respect to sterols are and address the importance of sampling strategies. From a pilot study it is concluded that differentiation between high and low metabolizers is independent of the type of sampling and constant over several days.