|An increasing number of studies are utilizing saliva sampling as a method for assessing the secretion of steroids. Saliva samples have certain advantages over plasma, being non-invasive and easily collected. Here we describe an analytical strategy that incorporates an on-line sample cleanup interfaced to tandem mass spectrometry that minimizes the sample preparation and guarantees low level detections. The protocol is fast, cheap, and amenable for large-scale studies. The total analysis time is less than 7 minutes, with a sample consumption of 50 µL and with a LOQ of 50 pM for cortisol and testosterone, and 15 pM for androstenedione.