Treatment of congenital adrenal hyperplasia (CAH) patients with glucocorticoids is often challenging since there is a delicate balance between over- and undertreatment. Treatment can be monitored non-invasively by measuring salivary androstenedione (A4) and 17-hydroxyprogesterone (17-OHP). Optimal treatment monitoring requires establishment of reference values in saliva. For this purpose, we developed a sensitive LC-MS/MS method, assessed salivary A4 and 17-OHP stability and measured A4 and 17-OHP concentrations in saliva collected from 255 healthy paediatric and adult volunteers in the morning, afternoon and evening. We demonstrated a diurnal rhythm and age-dependency for A4 and 17-OHP in saliva. These steroids remain stable in saliva at ambient temperature, which enables saliva collection by the patient at home. For effective interpretation, considering the time-point of saliva sampling in combination with corresponding age-adjusted reference values is necessary.