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Abstract: Background: Salivary steroid measurements have the potential to provide a non-invasive assessment of serum free steroid concentrations. Salivary testing has been proposed as a possible substitute for serum testing partly because of easy sample collection. Methods: We developed a liquid chromatography–tandem mass spectrometry (LC–MS/MS) assay for simultaneous measurement of testosterone and androstendione in saliva. We spiked 100μL of saliva with isotope-labeled internal standards and performed extraction with diethyl ether. After evaporation both steroids were analyzed by LC–MS/MS operating in the positive mode electrospray ionization (ESI) after separation on a reversed-phase column. Results: The calibration curves for analysis of testosterone and androstendione exhibited consistent linearity and reproducibility in the range of 20–1000pmol/L. The limit of quantitation (LOQ) was 10pmol/L for both steroids. The mean recovery of the analytes added to saliva ranged from 97 to 102%. Between-run CVs were 3.6–4.0% for testosterone and 4.8–8.0% for androstendione. The upper limit (the 97.5 percentile) of testosterone and androstendione reference ranges for males was 356pmol/L and 344pmol/L and that for females 39pmol/L and 349pmol/L, respectively. Conclusions: This LC–MS/MS method has the required lower limit of quantification for analysis of salivary concentrations of testosterone and androstendione. The assay is rapid, reliable and simple to perform with a routine LC–MS/MS spectrometer with required sensitivity. [Copyright &y& Elsevier]