In recent years measurement of steroid hormones like testosterone, androstenedione, cortisol, and cortisone has become increasingly important in both patient care and research. This is mainly due to the simple and non-invasive sample collection. We investigated in twenty healthy volunteers whether there is a difference between steroid hormone concentrations in unstimulated and parotid gland saliva as well as stimulated saliva collected while chewing without aid, using cotton and synthetic Salivettes, citric acid, or chewing gum. Testosterone, androstenedione, cortisol, and cortisone were measured in all saliva samples using isotope dilution liquid chromatographytandem mass spectrometry (ID-LCMS/MS). Testosterone, androstenedione, and cortisol concentrations were not affected by stimulation by chewing itself, whereas cortisone levels decreased (mean decrease was 11%). Levels of all four hormones were lower in parotid gland saliva compared to unstimulated saliva (mean decrease was 27, 14, 24, and 27% respectively). Salivary levels of testosterone, androstenedione, and cortisone decreased when using synthetic Salivettes (mean decrease was 56, 38, and 25% respectively) and increased when using cotton Salivettes (mean increase was 238, 50, and 34% respectively) compared to chewing without aid, whereas cortisol levels in saliva were unaffected by both types of Salivettes. Citric acid stimulation decreased salivary cortisone levels (mean decrease was 35%). Chewing gum decreased cortisone levels (mean decrease was 25%). In conclusion, the way saliva is collected should be taken into account when analysing and interpreting salivary hormone concentrations. Parotid gland saliva has lower steroid hormone levels than total saliva. Stimulation on its own does not change the concentrations of testosterone, androstenedione, and cortisol, but decreased the levels of cortisone. This is probably due to the rate-limited conversion of cortisol to cortisone in salivary glands. Stimulation with Salivettes, should be avoided for analysis of testosterone, androstenedione, and cortisone. Stimulation with citric acid and chewing gum are not suitable for analysis of cortisone.