Measuring circulating androgen and gestagen concentrations is essential for the diagnosis and treatment monitoring of pathological conditions caused by abnormal steroidogenesis, such as congenital adrenal hyperplasia (CAH) and polycystic ovary syndrome (PCOS). Saliva collection represents a simple and non-invasive technique advantageous for multi sample profiling. We therefore developed a liquid chromatography-tandem mass spectrometry assay for the simultaneous quantification of progesterone, 17-hydroxyprogesterone, classic pathway androgens (testosterone, androstenedione) and 11-oxygenated pathway androgens (11-hydroxyandrostenedione, 11-ketotestosterone). 11-oxygenated androgens have recently been show to represent the major androgens in CAH and PCOS and 11-ketotestosterone activates the androgen receptor with similar potency to testosterone. Samples (300 μL) were prepared by supported liquid extraction with dichloromethane. Online automated solid phase extraction (Waters OSM) of the reconstituted extracts was performed using C18-cartridges prior to LC on a C8-column with a water/methanol gradient. Quantification was performed with a Waters TQ-S mass spectrometer. Total run-time injection-to-injection was 6.4 minutes. The assay was validated for sensitivity, specificity, recovery, matrix effects, intra- and interassay imprecision, post-extraction stability, carry-over and dilution linearity. All parameters were acceptable according to the FDA Guidance for Industry: Bioanalytical Method Validation. Applicability of the assay was demonstrated by determining diurnal and menstrual alterations for three healthy female volunteers.