Background Saliva samples are easily collectable and non-invasive, and the monitoring of natural steroidal hormones, such as estrone (E1), 17β-estradiol (E2), estriol (E3), progesterone (P), and testosterone (T), in saliva has attracted much attention due to its numerous potential clinical and health-related applications. Because E1, E2, E3, P and T are useful indicators in numerous clinical and health-related diagnoses, there is a need for simultaneous determination. Results A gas chromatography-mass spectrometric assay was developed for rapid simultaneous determination of E1, E2, E3, P and T in saliva for clinical diagnoses. Extraction was achieved with a liquid extraction using 3.0 mL of pentane. The extract was dried and silylated with N-methyl-N-(trimethylsilyl) trifluoroacetamide/NH4I (100:2) under a catalysis of 1.5% dithioerythritol for 10 min at 90°C. The accuracy of the analytes was in the range of 96% to 112% at concentrations of 0.05 and 0.10 μg/L (5.0 and 10.0 μg/L for E3), respectively, with relative standard deviations of less than 11%. The lowest quantification limits were from 0.002 to 0.6 μg/L for 1.0 mL of saliva. Conclusion Natural steroidal hormones were detected in the concentration ranges of nd to 0.2 μg/L in human saliva. The salivary testosterone values in the patients with prostatic carcinoma were significantly lower than in normal males. The method may useful in numerous clinical and health-related diagnoses.