Recent studies showed that non-hormonal supplements such as vitamins, minerals and amino acids can contain anabolic androgenic steroids not declared on the labels of the products. These undeclared substances (often prohormones of testosterone or 19-nortestosterone) can cause health risks to consumers and might lead to positive results in sports doping control, especially for the nandrolone metabolite norandrosterone. The analysis of nutritional supplements for anabolic steroids has proven to be rather difficult due to the different matrices in the various products. To conduct a broad-based analysis, a few robust methods capable of analysing various matrices are needed. To obtain a sensitive gas chromatography-mass spectrometry (GC-MS) analysis, a method including extraction and purification of the analytes followed by GC-MS analysis of the trimethylsilyl (TMS) derivatives of the steroids was developed. The limit of detection was improved by the addition of a mixture of 1-N,N-diisopropylamino-n-alkanes (DIPAs) to the final extract. In pure creatine monohydrate powder the limits of detection were demonstrated to be 0.1 ng μg −1 for dehydroepiandrosterone (DHEA) and estr-4-ene-3β,17β-diol, 0.7 ng g−1 for 5α-androstane-3β,17β-diol and androsta-1,4-diene-3,17-dione, 1 ng g−1 for estr-5-ene-3β,17β-diol, estr-4-ene-3,17-dione, 19-nortestosterone, androst-4-ene-3, 17-dione and testosterone, and 2 ng g−1 for androst-4-ene-3β,17β-diol and androst-5-ene-3β,17β-diol. The recovery (determined at 200 ng g−1) ranged from 32% for 19-nortestosterone to 92% for androst-5-ene-3β,17β-diol. During the investigation of different nutritional supplements, several analytical difficulties occurred. Aspects of homogenization, extraction, separation, derivatization and GC-MS measurement as well as strategies for the solution of problems arising were optimized. For quantitative measurements of the steroids in nutritional supplements, deuterated internal standards of the specific steroids or standard addition are necessary to compensate for matrix effects.